Mutant hydroxyphenylpyruvate dioxygenase polypeptides and methods of use

ABSTRACT

Compositions and methods for conferring hydroxyphenyl pyruvate dioxygenase (HPPD) herbicide resistance or tolerance to plants are provided. Compositions include amino acid sequences, and variants and fragments thereof, for mutant HPPD polypeptides. Nucleic acids that encode the mutant HPPD polypeptides are also provided. Methods for conferring herbicide resistance or tolerance, particularly resistance or tolerance to certain classes of herbicides that inhibit HPPD, in plants are further provided. Methods are also provided for selectively controlling weeds in a field at a crop locus and for the assay, characterization, identification and selection of the mutant HPPDs of the current invention that provide herbicide tolerance.

RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application No. 61/224,661,filed Jul. 10, 2009, and to U.S. Provisional Application No. 61/146,513,filed Jan. 22, 2009, which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to mutant hydroxyphenyl pyruvatedioxygenase (HPPD) polypeptides that confer herbicide resistance ortolerance to plants and the nucleic acid sequences that encode them.Methods of the invention relate to the production and use of plants thatexpress these mutant HPPD polypeptides and that are resistant to HPPDherbicides.

BACKGROUND OF THE INVENTION

The hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes that catalyzethe reaction in which para-hydroxyphenylpyruvate (HPP) is transformedinto homogentisate. This reaction takes place in the presence ofenzyme-bound iron (Fe²⁺) and oxygen. Herbicides that act by inhibitingHPPD are well known, and include isoxazoles, diketonitriles, triketones,and pyrazolinates (Hawkes “Hydroxyphenylpyruvate Dioxygenase (HPPD)—TheHerbicide Target.” In Modern Crop Protection Compounds. Eds. Krämer andSchirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp. 211-220).Inhibition of HPPD blocks the biosynthesis of plastoquinone (PQ) fromtyrosine. PQ is an essential cofactor in the biosynthesis of carotenoidpigments which are essential for photoprotection of the photosyntheticcentres. HPPD-inhibiting herbicides are phloem-mobile bleachers whichcause the light-exposed new meristems and leaves to emerge white. In theabsence of carotenoids, chlorophyll is photo-destroyed and becomesitself an agent of photo-destruction via the photo-generation of singletoxygen.

Methods are also known for providing plants that are tolerant to HPPDherbicides and have included: 1) overexpressing the HPPD enzyme so as toproduce quantities of HPPD enzyme in the plant that are sufficient inrelation to a given herbicide so as to have enough of the functionalenzyme available despite the presence of its inhibitor; and 2) mutatingthe target HPPD enzyme into a functional HPPD that is less sensitive toherbicides. With respect to mutant HPPDs, while a given mutant HPPDenzyme may provide a useful level of tolerance to some HPPD-inhibitorherbicides, the same mutant HPPD may be quite inadequate to providecommercial levels of tolerance to a different, more desirableHPPD-inhibitor herbicide (See, e.g., U.S. App. Pub. No. 2004/0058427;and PCT App. Pub. Nos. WO 98/20144 and WO 02/46387; see also U.S. App.Pub. No. 2005/0246800 relating to identification and labelling ofsoybean varieties as being relatively HPPD tolerant). For example,HPPD-inhibitor herbicides may differ in terms of the spectrum of weedsthey control, their manufacturing cost, and their environmentalbenefits.

Accordingly, new methods and compositions for conferring HPPD herbicidetolerance upon various crops and crop varieties are needed.

SUMMARY OF THE INVENTION

Compositions and methods for conferring hydroxyphenyl pyruvatedioxygenase (HPPD) herbicide resistance or tolerance to plants areprovided. The compositions include nucleotide and amino acid sequencesfor mutant HPPD polypeptides. The polypeptides of the invention aremutant HPPDs that have HPPD enzymatic activity and that conferresistance or tolerance in plants to certain classes of herbicides thatinhibit HPPD. In one embodiment, the compositions of the inventioncomprise a mutant HPPD polypeptide having at least 80% sequence identityto SEQ ID NO:27, where the polypeptide has HPPD enzymatic activity, andwhere the polypeptide contains one or more amino acid additions,substitutions, or deletions selected from the group consisting of:

1) R(K,A,R)SQI(Q,E)T (SEQ ID NO:28), wherein the first Q is replacedwith any other amino acid, particularly with A, G, M, T, S, C, R, F andmore particularly with P;

2) R(K,A,R)SQI(Q,E)T (SEQ ID NO:28), wherein I is replaced with anyother amino acid, particularly with V, S, A, P, T, L or G;

3) (P,A,S)G(V,L)QH(I,L,M) (SEQ ID NO:29), wherein Q is replaced with anyother amino acid, particularly with N, R, G, A, S, T, E or C, and moreparticularly with A or H;

4) G(I,V) LVD(R,K)D (SEQ ID NO:30), wherein L is replaced with any otheramino acid, particularly with M or A;

5) ESGLN(S,G) (SEQ ID NO:31), wherein L is replaced with any other aminoacid, particularly with M, H, G, F, C or I, and more particularly withM;

6) F(A,S)EF(T,V) (SEQ ID NO:32), wherein (A,S) is replaced with anyamino acid, particularly with W, G, M, F, Y or H;

7) G(I,V) LVD(R,K)D (SEQ ID NO:30) and ESGLN(S,G) (SEQ ID NO:31), whereL in both sequences is replaced with M;

8) EVELYGDVV (SEQ ID NO:37), wherein Y is replaced with any other aminoacid, particularly with D, V, E, K, or A;

9) RFDHVVGNV (SEQ ID NO:38), wherein the first V is replaced with anyother amino acid; such as I, A, M, or C;

10) DHVVGNVPE (SEQ ID NO:39), wherein G is replaced with any other aminoacid; such as H or C;

11) HVVGNVPEM (SEQ ID NO:40), wherein N is replaced with any other aminoacid; such as C;

12) NVPEMAPVI (SEQ ID NO:41), wherein M is replaced with any other aminoacid; such as L;

13) GFHEFAEFT (SEQ ID NO:42), wherein F is replaced with any other aminoacid; such as M, I, or L;

14) GTTESGLNS (SEQ ID NO:43), wherein S is replaced with any other aminoacid; such as T;

15) TTESGLNSV (SEQ ID NO:44), wherein G is replaced with any other aminoacid; such as R, S, or A;

16) ESGLNSVVL (SEQ ID NO:45), wherein N is replaced with any other aminoacid; such as R or M;

17) GLNSVVLAN (SEQ ID NO:46), wherein the first V is replaced with anyother amino acid; such as M, I, A, or K;

18) LNSVVLANN (SEQ ID NO:47), wherein V is replaced with any other aminoacid; such as I;

19) SEAVLLPLN (SEQ ID NO:48), wherein L is replaced with any other aminoacid; such as V or K;

20) EAVLLPLNE (SEQ ID NO:49), wherein L is replaced with any other aminoacid; such as M or F;

21) VLLPLNEPV (SEQ ID NO:50), wherein the third L is replaced with anyother amino acid; such as I, M, or V;

22) LLPLNEPVH (SEQ ID NO:51), wherein N is replaced with any other aminoacid; such as A;

23) HGTKRRSQI (SEQ ID NO:52), wherein R is replaced with any other aminoacid; such as G;

24) SQIQTYLEY (SEQ ID NO:53), wherein T is replaced with any other aminoacid; such as E;

25) QIQTYLEYH (SEQ ID NO:54), wherein Y is replaced with any other aminoacid; such as F;

26) GVQHIALAS (SEQ ID NO:55), wherein I is replaced with any other aminoacid; such as M, L, or V;

27) GFEFMAPPQ (SEQ ID NO:57), wherein M is replaced with any other aminoacid; such as Q or L;

28) FEFMAPPQA (SEQ ID NO:58), wherein the first A is replaced with anyother amino acid; such as S, P, D, R, N, Y, K, or H;

29) FMAPPQAKY (SEQ ID NO:59), wherein P is replaced with any other aminoacid; such as A or R;

30) QAKYYEGVR (SEQ ID NO:60), wherein Y is replaced with any other aminoacid; such as K, R, D, Q, or E;

31) GVRRIAGDV (SEQ ID NO:61), wherein I is replaced with any other aminoacid; such as R or L;

32) VLLQIFTKP (SEQ ID NO:62), wherein I is replaced with any other aminoacid; such as V;

33) LLQIFTKPV (SEQ ID NO:63), wherein F is replaced with any other aminoacid; such as L;

34) LQIFTKPVG (SEQ ID NO:64), wherein T is replaced with any other aminoacid; such as S, P, D, R, N, Y, or H;

35) IFTKPVGDR (SEQ ID NO:65), wherein P is replaced with any other aminoacid; such as N;

36) RPTFFLEMI (SEQ ID NO:66), wherein F is replaced with any other aminoacid; such as L;

37) FLEMIQRIG (SEQ ID NO:67), wherein I is replaced with any other aminoacid; such as V or C;

38) GGCGGFGKG (SEQ ID NO:68), wherein the fourth G is replaced with anyother amino acid; such as A, S, or T;

39) GGFGKGNFS (SEQ ID NO:69), wherein K is replaced with any other aminoacid; such as L, A, E, or V;

40) GFGKGNFSE (SEQ ID NO:70), wherein G is replaced with any other aminoacid; such as I;

41) FGKGNFSEL (SEQ ID NO:71), wherein N is replaced with any other aminoacid; such as I;

42) KGNFSELFK (SEQ ID NO:72), wherein S is replaced with any other aminoacid; such as N, G, K, or Q;

43) GNFSELFKS (SEQ ID NO:73), wherein E is replaced with any other aminoacid; such as Q;

44) ELFKSIEDY (SEQ ID NO:74), wherein S is replaced with any other aminoacid; such as A;

45) LFKSIEDYE (SEQ ID NO:75), wherein I is replaced with any other aminoacid; such as L or F;

46) HVVGNVPEM (SEQ ID NO:40), wherein N is replaced with any other aminoacid, particularly a C, and the amino acid sequence ELGVLVDRD (SEQ IDNO:76), wherein the second L is replaced with any other amino acid,particularly an M;

47) LNSVVLANN (SEQ ID NO:47), wherein the second V is replaced with anyother amino acid, particularly an I, and the amino acid sequenceELGVLVDRD (SEQ ID NO:76), wherein the second L is replaced with anyother amino acid, particularly an M;

48) VLLPLNEPV (SEQ ID NO:50), wherein the third L is replaced with anyother amino acid, particularly an M, and the amino acid sequenceVLLQIFTKP (SEQ ID NO:62), wherein I is replaced with any other aminoacid, particularly a V;

49) GGCGGFGKG (SEQ ID NO:68), wherein the fourth G is replaced with anyother amino acid, particularly a T, and the amino acid sequenceELGVLVDRD (SEQ ID NO:76), wherein the second L is replaced with anyother amino acid, particularly an M;

50) FHEFAEFTAED (SEQ ID NO:76), wherein the first A, the second E, andthe second F are replaced with any other amino acid, particularly wherethe A is replaced with an S or a W, the E is replaced with a T, and/orthe F is replaced with an A or a V;

51) HGTKRRSQIQ (SEQ ID NO:77), wherein the first R is replaced with anyother amino acid, particularly with a K, and the second R is deleted;

52) GTKRRSQIQ (SEQ ID NO:78), wherein the second R is deleted;

53) FMAPPQAKY (SEQ ID NO:59), wherein the second P is deleted;

54) GNFSELFKS (SEQ ID NO:73), wherein the E is deleted;

55) GVRRIAGDV (SEQ ID NO:61), wherein the I is deleted;

56) DQGVLLQIFTKP (SEQ ID NO:79), wherein the first L and the I arereplaced with any other amino acid, particularly where the A is replacedwith an M and/or the I is replaced with an L;

57) GKGNFSELFK (SEQ ID NO:80), wherein the F and the S are replaced withany other amino acid, particularly where the F is replaced with a Gand/or the S is replaced with an A;

58) KGNFSELFKS (SEQ ID NO:56), wherein the first S and the E arereplaced with any other amino acid, particularly where the S is replacedwith an N, G, or K and/or the E is replaced with an S or an A;

59) GGCGGFGKG (SEQ ID NO:68) wherein the K is replaced with any otheramino acid, such as T, S, Q, L, A, I, H, E, G, M, C or V, preferably T;

60) GGCGGFGKG (SEQ ID NO:68), wherein the sixth G is replaced with anyother amino acid, such as R, E, D, H, M, F, W, N, or C, preferably H orC;

61) ESGLN(S,G) (SEQ ID NO:31), wherein the first G is replaced with anyother amino acid, particularly with R, S, or A; and

62) VLLPLNEPV (SEQ ID NO:50), wherein the second L is replaced with anyother amino acid, such as M, F, or V.

In another embodiment, the compositions of the invention comprise amutant HPPD polypeptide having at least 80% sequence identity to SEQ IDNO:14 or to SEQ ID NO:27, where the polypeptide has HPPD enzymaticactivity, and where the polypeptide contains one or more amino acidsubstitutions selected from the group consisting of:

1) R(K,A,R)SQI(Q,E)T (SEQ ID NO:28), wherein I is replaced with anyother amino acid, particularly with V, S, A, P, T, L or G;

2) (P,A,S)G(V,L)QH(I,L,M) (SEQ ID NO:29), wherein Q is replaced with anyother amino acid, particularly with N, R, G, A, S, T, E or C, and moreparticularly with A or H;

3) G(I,V) LVD(R,K)D (SEQ ID NO:30), wherein L is replaced with any otheramino acid, particularly with M or A;

4) ESGLN(S,G) (SEQ ID NO:31), wherein L is replaced with any other aminoacid, particularly with M, H, G, F, C or I, and more particularly withM;

5) F(A,S)EF(T,V) (SEQ ID NO:32), wherein (A,S) is replaced with anyamino acid, particularly with W, G, M, F, Y or H;

6) RFDHVVGNV (SEQ ID NO:38), wherein the first V is replaced with anyother amino acid, such as I, A, M, or C;

7) GLNSVVLAN (SEQ ID NO:46), wherein the first V is replaced with anyother amino acid, such as M, I, A, or K;

8) VLLPLNEPV (SEQ ID NO:50), wherein the third L is replaced with anyother amino acid, such as I, M, or V;

9) GFEFMAPPQ (SEQ ID NO:57), wherein M is replaced with any other aminoacid, such as Q or L;

10) FEFMAPPQA (SEQ ID NO:58), wherein the first A is replaced with anyother amino acid, such as S, P, D, R, N, Y, K, or H;

11) GGCGGFGKG (SEQ ID NO:68), wherein the fourth G is replaced with anyother amino acid, such as A, S, or T;

12) GGCGGFGKG (SEQ ID NO:68) wherein the K is replaced with any otheramino acid, such as T, S, Q, L, A, I, H, E, G, M, C or V, preferably T;

13) GGCGGFGKG (SEQ ID NO:68), wherein the sixth G is replaced with anyother amino acid, such as R, E, D, H, M, F, W, N, or C, preferably H orC;

14) ESGLN(S,G) (SEQ ID NO:31), wherein the first G is replaced with anyother amino acid, particularly with R, S, or A; and

15) VLLPLNEPV (SEQ ID NO:50), wherein the second L is replaced with anyother amino acid, such as M, F, or V.

Exemplary mutant HPPD polypeptides according to the invention correspondto the amino acid sequences set forth in SEQ ID NOS:14-26, and variantsand fragments thereof. Nucleic acid molecules comprising polynucleotidesequences that encode the mutant HPPD polypeptides of the invention arefurther provided, e.g., SEQ ID NOS:1-13. Compositions also includeexpression cassettes comprising a promoter operably linked to anucleotide sequence that encodes a mutant HPPD polypeptide of theinvention, alone or in combination with one or more additional nucleicacid molecules encoding polypeptides that confer desirable traits.Transformed plants, plant cells, and seeds comprising an expressioncassette of the invention are further provided.

The compositions of the invention are useful in methods directed toconferring herbicide resistance or tolerance to plants, particularlyresistance or tolerance to certain classes of herbicides that inhibitHPPD. In particular embodiments, the methods comprise introducing into aplant at least one expression cassette comprising a promoter operablylinked to a nucleotide sequence that encodes a mutant HPPD polypeptideof the invention. As a result, the mutant HPPD polypeptide is expressedin the plant, and the mutant HPPD is less sensitive to HPPD-inhibitingherbicides, thereby leading to resistance or tolerance toHPPD-inhibiting herbicides.

Methods of the present invention also comprise selectively controllingweeds in a field at a crop locus. In one embodiment, such methodsinvolve over-the-top pre- or postemergence application ofweed-controlling amounts of HPPD herbicides in a field at a crop locusthat contains plants expressing the mutant HPPD polypeptides of theinvention. In other embodiments, methods are also provided for theassay, characterization, identification, and selection of the mutantHPPDs of the current invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Km and Vmax values of the Avena-derived HPPD polypeptidecorresponding to the amino acid sequence set forth in SEQ ID NO:14.

FIGS. 2A-2B show on rate (FIG. 2A) and off rate (FIG. 2B) determinationsfor a complex of structure B with the HPPD polypeptide corresponding tothe amino acid sequence set forth in SEQ ID NO:14.

FIG. 3 shows an off rate determination for a complex of structure D withthe HPPD polypeptide corresponding to the amino acid sequence set forthin SEQ ID NO:14.

FIGS. 4A-4C show off rate determinations at ice temperature forcomplexes of structure B with the HPPD polypeptides corresponding to theamino acid sequences set forth in SEQ ID NO:14 (FIG. 4A), 24 (FIG. 4B),and 26 (FIG. 4C).

FIG. 5 shows mesotrione inhibition of pyomelanin formation by E. coliBL21 expressing different variants of HPPD. Left bar=(error range forn=3) average A 430 nm with zero mesotrione present in the medium andright bar=(n=3) average A 430 nm with 12.5 ppm present in the medium.Control is pET24 empty vector where no HPPD is expressed.

FIG. 6 shows a representation of binary vector 17146 for soybeantransformation, conferring HPPD resistance with a soybean codonoptimized Oat HPPD gene encoding SEQ ID NO:24. This binary vector alsocontains double PAT selectable markers for glufosinate selection.

FIG. 7 shows a representation of binary vector 17147 for soybeantransformation conferring HPPD resistance with a soybean codon optimizedOat HPPD gene encoding SEQ ID NO:24 and also conferring tolerance toglyphosate (selectable marker).

FIG. 8 shows a representation of binary vector 15764 containing asoybean codon optimized Oat HPPD gene (encoding SEQ ID NO:14) driven bythe TMV omega enhancer and a TATA box.

FIG. 9 shows a representation of binary vector 17149 for soybeantransformation conferring tolerance to HPPD herbicides and toglufosinate, containing an expression cassette expressing an HPPDvariant (SEQ ID NO:26) along with two PAT gene cassettes.

FIGS. 10A-10D depict the time-dependence of inhibition of a mutant ofHPPD (G408A) by herbicide compounds B (FIGS. 10A-10B) and C (FIGS.10C-10D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods directed toconferring hydroxyphenyl pyruvate dioxygenase (HPPD) herbicideresistance or tolerance to plants. Compositions include amino acidsequences for mutant HPPD polypeptides having HPPD enzymatic activity,and variants and fragments thereof. Nucleic acids that encode the mutantHPPD polypeptides of the invention are also provided. Methods forconferring herbicide resistance or tolerance to plants, particularlyresistance or tolerance to certain classes of herbicides that inhibitHPPD, are further provided. Methods are also provided for selectivelycontrolling weeds in a field at a crop locus and for the assay,characterization, identification and selection of the mutant HPPDs ofthe current invention that provide herbicide tolerance.

Within the context of the present invention the terms hydroxy phenylpyruvate dioxygenase (HPPD), 4-hydroxy phenyl pyruvate dioxygenase(4-HPPD) and p-hydroxy phenyl pyruvate dioxygenase (p-HPPD) aresynonymous.

“HPPD herbicides” are herbicides that are bleachers and whose primarysite of action is HPPD. Many are well known and described elsewhereherein and in the literature (Hawkes “Hydroxyphenylpyruvate Dioxygenase(HPPD)—The Herbicide Target.” In Modern Crop Protection Compounds. Eds.Krämer and Schirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp.211-220; Edmunds “Hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitors:Triketones.” In Modern Crop Protection Compounds. Eds. Krämer andSchirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp. 221-242). Asused herein, the term “HPPD herbicides” refers to herbicides that acteither directly or indirectly to inhibit HPPD, where the herbicides arebleachers, and where inhibition of HPPD is at least part of theherbicide's mode of action on plants.

As used herein, plants which are substantially “tolerant” to a herbicideexhibit, when treated with said herbicide, a dose/response curve whichis shifted to the right when compared with that exhibited by similarlysubjected non tolerant like plants. Such dose/response curves have“dose” plotted on the x-axis and “percentage kill or damage”,“herbicidal effect” etc. plotted on the y-axis. Tolerant plants willtypically require at least twice as much herbicide as non tolerant likeplants in order to produce a given herbicidal effect. Plants which aresubstantially “resistant” to the herbicide exhibit few, if any,necrotic, lytic, chlorotic or other lesions or, at least, none thatimpact significantly on yield, when subjected to the herbicide atconcentrations and rates which are typically employed by theagricultural community to kill weeds in the field.

As used herein, “non-transgenic-like plants” are plants that are similaror the same as transgenic plants but that do not contain a transgeneconferring herbicide resistance.

As used herein, the term “confer” refers to providing a characteristicor trait, such as herbicide tolerance or resistance and/or otherdesirable traits to a plant.

As described elsewhere herein, the term “heterologous” means fromanother source. In the context of DNA, “heterologous” refers to anyforeign “non-self” DNA including that from another plant of the samespecies. For example, in the present application a soybean HPPD genethat was transgenically expressed back into a soybean plant would stillbe described as “heterologous” DNA.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element. Throughout thespecification the word “comprising,” or variations such as “comprises”or “comprising,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps.

A variety of additional terms are defined or otherwise characterizedherein.

HPPD Sequences

The compositions of the invention include isolated or substantiallypurified mutant HPPD polynucleotides and polypeptides as well as hostcells comprising mutant HPPD polynucleotides. Specifically, the presentinvention provides mutant HPPD polypeptides that have HPPD enzymaticactivity and that confer resistance or tolerance in plants to certainclasses of herbicides that inhibit HPPD, and variants and fragmentsthereof. Nucleic acids that encode the mutant HPPD polypeptides of theinvention are also provided.

Mutant HPPD polypeptides of the presenting invention have amino acidchanges at one or more positions relative to the starting wild typesequence from which they are derived, and exhibit enhanced tolerance toone or more HPPD inhibitor herbicides. HPPD enzymes that exhibitenhanced tolerance to an HPPD herbicide may do so by virtue ofexhibiting, relative to the like unmutated starting enzyme:

a) a lower Km value for the natural substrate, 4-hydroxyphenylpyruvate;

b) a higher kcat value for converting 4-hydroxyphenylpyruvate tohomogentisate;

c) a lower value of the rate constant, kon, governing formation of anenzyme:

HPPD inhibitor herbicide complex;

d) an increased value of the rate constant, koff, governing dissociationof an enzyme: HPPD inhibitor herbicide complex; and/or

e) as a result of changes in one or both of c) and d), an increasedvalue of the equilibrium constant, Ki (also called Kd), governingdissociation of an enzyme: HPPD inhibitor herbicide complex. DNAsequences encoding such improved mutated HPPDs are used in the provisionof HPPD plants, crops, plant cells and seeds of the current inventionthat offer enhanced tolerance or resistance to one or more HPPDherbicides as compared to like plants likewise expressing the unmutatedstarting enzyme.

Increases in the value of koff are of particular value in improving theability of HPPD to confer resistance to a HPPD herbicide. As oneexample, compounds B and C exhibit similar Kd values with respect to theHPPD variant of SEQ ID NO:14 but differ in that the koff value forcompound B is about 10-fold greater as compared to the koff value forcompound C, and plants expressing SEQ ID NO:14 show superior resistanceto compound B than to compound C.

Site-directed mutations of genes encoding plant-derived HPPDs areselected so as to encode amino acid changes selected from the list beloweither singly or in combination. Genes encoding such mutant forms ofplant HPPDs are useful for making crop plants resistant to herbicidesthat inhibit HPPD. Plant HPPD genes so modified are especially suitablefor use in transgenic plants in order to confer herbicide tolerance orresistance upon crop plants.

Many HPPD sequences are known in the art and can be used to generatemutant HPPD sequences by making the corresponding amino acidsubstitutions, deletions, and additions described herein. The HPPD aminoacid sequence of Avena sativa is set forth in SEQ ID NO:27. A singledeletion variant of the Avena sativa HPPD is set forth in SEQ ID NO:14.Thus, a known or suspected HPPD sequence can be aligned with, forexample, SEQ ID NO:14 or SEQ ID NO:27 using standard sequence alignmenttools, and the corresponding amino acid substitutions, deletions, and/oradditions described herein with respect to SEQ ID NO:14 or to SEQ IDNO:27 can be made in the reference sequence.

In one embodiment, the compositions of the invention comprise a mutantHPPD polypeptide having at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO:27 (the HPPD amino acid sequence ofAvena sativa) or where the HPPD amino acid sequence derives from aplant, where the polypeptide has HPPD enzymatic activity, and where thepolypeptide contains one or more amino acid sequence additions,substitutions, or deletions corresponding to the amino acid positionslisted in column 1 of Table 1, optionally in further combination withknown mutations (see e.g., WO2009/144079). In various embodiments, anamino acid at one or more position(s) listed in column 1 of Table 1 isreplaced with any other amino acid. In another embodiment, thepolypeptide comprises one or more amino acid substitutions, additions,or deletions corresponding to the amino acid substitutions or additionslisted in column 2 of Table 1. In yet another embodiment, thepolypeptide comprises one or more substitutions corresponding to aconservative variant of the amino acids listed in column 2 of Table 1.For example, the polypeptide may comprise a mutation corresponding toamino acid position 217 of SEQ ID NO:14 (amino acid position 218 of SEQID NO:27), wherein that amino acid is replaced with alanine or aconservative substitution of alanine; or the polypeptide may comprise amutation corresponding to amino acid position 241 of SEQ ID NO:14 (aminoacid position 242 of SEQ ID NO:27), wherein that amino acid is replacedwith tryptophan or a conservative substitution of tryptophan; or thepolypeptide may comprise a mutation corresponding to amino acid position408 of SEQ ID NO:14 (amino acid position 409 of SEQ ID NO:27), whereinthat amino acid is replaced with alanine or a conservative substitutionof alanine. In particular embodiments, the amino acid sequence of themutant HPPD polypeptide of the invention is selected from the groupconsisting of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26.

TABLE 1 Exemplary HPPD Mutations Mutable amino acid position relative toSEQ ID NO: 14 Substitution, addition, or deletion* 172 D, V, E, K, or A217 I, A, M, or C 219 H or C 220 C 224 L 240 M, I, or L 241 S, W, G, M,F, Y, or H 244 V 253 T 254 R, S, or A 255 M, H, G, F, C, or I 256 R or M257 G 258 M, I, A, or K 259 I 268 V or K 269 M, F, or V 271 I, M, or V272 A 280 G or K 281 Delete R 281-282 insert K, A, or R between R282 andS283 284 V, S, A, P, T, L, or G 286 E 287 F 294 A or S 296 L 297 N, R,G, A, H, S, T, E, or C 299 L or M 299 M, L, or V 325 Q or L 326 K, S, P,D, R, N, Y, or H 328 A or R 328 Delete P 333 K, R, D, Q, or E 336 DeleteE 339 R or L 339 Delete I 357 I 358 L 358 M or A 361 K 367 M 370 V or L371 L 372 S, P, D, R, N, Y, or H 374 N 382 L 386 V or C 408 A, S, or T410 T, S, L, A, I, V, Q, H, E, G, M, C, V, or T 411 I 413 I 414 G 415 A,N, G, K, or Q 416 S, A, or Q 420 A *Unless otherwise denoted, the aminoacids listed in this column represent the potential substitutions at theindicated position.

In another embodiment, the compositions of the invention comprise amutant HPPD polypeptide having at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to SEQ ID NO:27 (the HPPD amino acidsequence of Avena sativa) or where the HPPD amino acid sequence derivesfrom a plant, where the polypeptide has HPPD enzymatic activity, andwhere the polypeptide contains one or more amino acid sequencesubstitutions corresponding to the amino acid positions listed in column1 of Table 2, optionally in further combination with known mutations(see e.g., WO2009/144079). In various embodiments, an amino acid at oneor more position(s) listed in column 1 of Table 2 is replaced with anyother amino acid. In another embodiment, the polypeptide comprises oneor more amino acid substitutions corresponding to the amino acidsubstitutions listed in column 2 of Table 2. In yet another embodiment,the polypeptide comprises one or more substitutions corresponding to aconservative variant of the amino acids listed in column 2 of Table 2.For example, the polypeptide may comprise a mutation corresponding toamino acid position 217 of SEQ ID NO:14 (amino acid position 218 of SEQID NO:27), wherein that amino acid is replaced with alanine or aconservative substitution of alanine; or the polypeptide may comprise amutation corresponding to amino acid position 241 of SEQ ID NO:14 (aminoacid position 242 of SEQ ID NO:27), wherein that amino acid is replacedwith tryptophan or a conservative substitution of tryptophan; or thepolypeptide may comprise a mutation corresponding to amino acid position408 of SEQ ID NO:14 (amino acid position 409 of SEQ ID NO:27), whereinthat amino acid is replaced with alanine or a conservative substitutionof alanine In particular embodiments, the amino acid sequence of themutant HPPD polypeptide of the invention is selected from the groupconsisting of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26.

TABLE 2 Exemplary HPPD Mutations Amino acid position (relative to SEQ IDNO: 14) Substitution 217 I, A, M, or C 241 S, W, G, M, F, Y, or H 254 R,S, or A 255 M, H, G, F, C, or I 258 M, I, A, or K 269 M, F, or V 271 M,I, or V 284 V, S, A, P, T, L, or G 297 N, R, G, S, T, E, C, A, or H 325Q or L 326 K, S, P, D, R, N, Y, or H 358 M or A 408 A, S, or T 411 T, S,L, A, I, Q, H, E, G, M, C, V, or T

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Polypeptides of the invention can be produced either from anucleic acid disclosed herein, or by the use of standard molecularbiology techniques. For example, a truncated protein of the inventioncan be produced by expression of a recombinant nucleic acid of theinvention in an appropriate host cell, or alternatively by a combinationof ex vivo procedures, such as protease digestion and purification.

Accordingly, the present invention also provides nucleic acid moleculescomprising polynucleotide sequences that encode mutant HPPD polypeptidesthat have HPPD enzymatic activity and that confer resistance ortolerance in plants to certain classes of herbicides that inhibit HPPD,and variants and fragments thereof. In general, the invention includesany polynucleotide sequence that encodes any of the mutant HPPDpolypeptides described herein, as well as any polynucleotide sequencethat encodes HPPD polypeptides having one or more conservative aminoacid substitutions relative to the mutant HHPD polypeptides describedherein. Conservative substitution tables providing functionally similaramino acids are well known in the art. The following five groups eachcontain amino acids that are conservative substitutions for one another:Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine(I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Asparagine (N), Glutamine (Q).

In one embodiment, the present invention provides a polynucleotidesequence encoding an amino acid sequence having at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:14 or to SEQID NO:27 or where the HPPD amino acid sequence derives from a plant,where the polypeptide has HPPD enzymatic activity, and where thepolypeptide contains one or more amino acid sequence additions,substitutions, or deletions as described herein. In particularembodiments, the polynucleotide sequence encodes a mutant HPPDpolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26. In another embodiment, the present invention provides apolynucleotide sequence selected from the group consisting of SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofinterfering enzyme activities and that is capable of being characterizedin respect of its catalytic, kinetic and molecular properties includesquite crude preparations of protein (for example recombinantly producedin cell extracts) having less than about 98%, 95% 90%, 80%, 70%, 60% or50% (by dry weight) of contaminating protein as well as preparationsfurther purified by methods known in the art to have 40%, 30%, 20%, 10%,5%, or 1% (by dry weight) of contaminating protein.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the mutant HPPDproteins can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatoften do not affect biological activity of the protein of interest maybe found in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found, Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

The polynucleotides of the invention can also be used to isolatecorresponding sequences from other organisms, particularly other plants.In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences based on their sequence homology to thesequences set forth herein.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art. See, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York).

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Methods for preparation of probes for hybridization and for constructionof cDNA and genomic libraries are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

By “hybridizing to” or “hybridizing specifically to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra, for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone nucleotide sequences that are homologues ofreference nucleotide sequences of the present invention: a referencenucleotide sequence preferably hybridizes to the reference nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.“Fragment” is intended to mean a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the mutant HPPD protein and hence haveHPPD enzymatic activity. Alternatively, fragments of a nucleotidesequence that are useful as hybridization probes or in mutagenesis andshuffling reactions to generate yet further HPPD variants generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence encoding the polypeptides of theinvention.

A fragment of a nucleotide sequence that encodes a biologically activeportion of a mutant HPPD protein of the invention will encode at least15, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 180, 200, 250,300, or 350 contiguous amino acids, or up to the total number of aminoacids present in a full-length mutant HPPD polypeptide of the invention.Fragments of a nucleotide sequence that are useful as hybridizationprobes or PCR primers generally need not encode a biologically activeportion of an HPPD protein.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide means having the entire nucleic acid sequence of a nativeor mutated HPPD sequence. “Native sequence” is intended to mean anendogenous sequence, i.e., a non-engineered sequence found in anorganism's genome.

Thus, a fragment of a nucleotide sequence of the invention may encode abiologically active portion of a mutant HPPD polypeptide, or it may be afragment that can be used as a hybridization probe etc. or PCR primerusing methods disclosed below. A biologically active portion of a mutantHPPD polypeptide can be prepared by isolating a portion of one of thenucleotide sequences of the invention, expressing the encoded portion ofthe mutant HPPD protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the mutant HPPDprotein. Nucleic acid molecules that are fragments of a nucleotidesequence of the invention comprise at least 15, 20, 50, 75, 100, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300contiguous nucleotides, or up to the number of nucleotides present in afull-length nucleotide sequence disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the referencepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the mutant HPPD polynucleotide. As used herein, a“reference” polynucleotide or polypeptide comprises a mutant HPPDnucleotide sequence or amino acid sequence, respectively. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Oneof skill in the art will recognize that variants of the nucleic acids ofthe invention will be constructed such that the open reading frame ismaintained. For polynucleotides, conservative variants include thosesequences that, because of the degeneracy of the genetic code, encodethe amino acid sequence of one of the mutant HPPD polypeptides of theinvention. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant polynucleotides also includesynthetically derived polynucleotide, such as those generated, forexample, by using site-directed mutagenesis but which still encode amutant HPPD protein of the invention. Generally, variants of aparticular polynucleotide of the invention will have at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, a polynucleotide that encodes apolypeptide with a given percent sequence identity to the polypeptidesof SEQ ID NOS: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26,are disclosed. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotides ofthe invention is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity across theentirety of the HPPD sequences described herein, i.e., when compared tothe full length HPPD sequences described herein.

“Variant” protein is intended to mean a protein derived from thereference protein by deletion or addition of one or more amino acids atone or more internal sites in the mutant HPPD protein and/orsubstitution of one or more amino acids at one or more sites in themutant HPPD protein. Variant proteins encompassed by the presentinvention are biologically active, that is they continue to possess thedesired biological activity of the mutant HPPD protein, that is, HPPDenzymatic activity and/or herbicide tolerance as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a mutant HPPDprotein of the invention will have at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity across the entirety of the amino acidsequence for the mutant HPPD protein as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a protein of the invention may differ from thatprotein by as few as 1-15 amino acid residues, as few as 1-10, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Methods of alignment of sequences for comparison are well known in theart and can be accomplished using mathematical algorithms such as thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; and the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of thesemathematical algorithms can be utilized for comparison of sequences todetermine sequence identity. Such implementations include, but are notlimited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin GeneticsSoftware Package, Version 10 (available from Accelrys Inc., 9685Scranton Road, San Diego, Calif., USA).

Gene Stacking

In certain embodiments the polynucleotides of the invention encodingmutant HPPD polypeptides or variants thereof that retain HPPD enzymaticactivity (e.g., a polynucleotide sequence encoding an amino acidsequence selected from the group consisting of SEQ ID NO:14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, and 26) can be stacked with anycombination of polynucleotide sequences of interest in order to createplants with a desired trait. A trait, as used herein, refers to thephenotype derived from a particular sequence or groups of sequences. Forexample, the polynucleotides encoding a mutant HPPD polypeptide orvariant thereof that retains HPPD enzymatic activity may be stacked withany other polynucleotides encoding polypeptides that confer a desirabletrait, including but not limited to resistance to diseases, insects, andherbicides, tolerance to heat and drought, reduced time to cropmaturity, improved industrial processing, such as for the conversion ofstarch or biomass to fermentable sugars, and improved agronomic quality,such as high oil content and high protein content.

Exemplary polynucleotides that may be stacked with polynucleotides ofthe invention encoding an mutant HPPD polypeptide or variant thereofthat retains HPPD enzymatic activity include polynucleotides encodingpolypeptides conferring resistance to pests/pathogens such as viruses,nematodes, insects or fungi, and the like. Exemplary polynucleotidesthat may be stacked with polynucleotides of the invention includepolynucleotides encoding: polypeptides having pesticidal and/orinsecticidal activity, such as other Bacillus thuringiensis toxicproteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514;5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), lectins(Van Damme et al. (1994) Plant Mol. Biol. 24:825, pentin (described inU.S. Pat. No. 5,981,722), and the like; traits desirable for disease orherbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat.No. 5,792,931); avirulence and disease resistance genes (Jones et al.(1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinoset al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants thatlead to herbicide resistance such as the S4 and/or Hra mutations;glyphosate resistance (e.g.,5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) gene, described inU.S. Pat. Nos. 4,940,935 and 5,188,642; or the glyphosateN-acetyltransferase (GAT) gene, described in Castle et al. (2004)Science, 304:1151-1154; and in U.S. Patent App. Pub. Nos. 20070004912,20050246798, and 20050060767)); glufosinate resistance (e.g.,phosphinothricin acetyl transferase genes PAT and BAR, described in U.S.Pat. Nos. 5,561,236 and 5,276,268); resistance to herbicides includingsulfonyl urea, DHT (2,4D), and PPO herbicides (e.g., glyphosate acetyltransferase, aryloxy alkanoate dioxygenase, acetolactate synthase, andprotoporphyrinogen oxidase); a cytochrome P450 or variant thereof thatconfers herbicide resistance or tolerance to, inter alia, HPPDherbicides (U.S. patent application Ser. No. 12/156,247; U.S. Pat. Nos.6,380,465; 6,121,512; 5,349,127; 6,649,814; and 6,300,544; and PCTPatent App. Pub. No. WO2007000077); and traits desirable for processingor process products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE), and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)); the disclosures of which are hereinincorporated by reference.

Thus, in one embodiment, the polynucleotides encoding a mutant HPPDpolypeptide or variant thereof that retains HPPD enzymatic activity arestacked with one or more polynucleotides encoding polypeptides thatconfer resistance or tolerance to an herbicide. In one embodiment, thedesirable trait is resistance or tolerance to an HPPD inhibitor. Inanother embodiment, the desirable trait is resistance or tolerance toglyphosate. In another embodiment, the desirable trait is resistance ortolerance to glufosinate.

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Plant Expression Cassettes

The compositions of the invention may additionally contain nucleic acidsequences for transformation and expression in a plant of interest. Thenucleic acid sequences may be present in DNA constructs or expressioncassettes. “Expression cassette” as used herein means a nucleic acidmolecule capable of directing expression of a particular nucleotidesequence in an appropriate host cell, comprising a promoter operativelylinked to the nucleotide sequence of interest (i.e., a polynucleotideencoding a mutant HPPD polypeptide or variant thereof that retains HPPDenzymatic activity, alone or in combination with one or more additionalnucleic acid molecules encoding polypeptides that confer desirabletraits) which is operatively linked to termination signals. It alsotypically comprises sequences required for proper translation of thenucleotide sequence. The coding region usually codes for a protein ofinterest but may also code for a functional RNA of interest, for exampleantisense RNA or a nontranslated RNA, in the sense or antisensedirection. The expression cassette comprising the nucleotide sequence ofinterest may be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular DNA sequence of the expressioncassette does not occur naturally in the host cell and must have beenintroduced into the host cell or an ancestor of the host cell by atransformation event. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter that initiates transcription only when thehost cell is exposed to some particular external stimulus. Additionally,the promoter can also be specific to a particular tissue or organ orstage of development.

The present invention encompasses the transformation of plants withexpression cassettes capable of expressing a polynucleotide of interest,i.e., a polynucleotide encoding a mutant HPPD polypeptide or variantthereof that retains HPPD enzymatic activity, alone or in combinationwith one or more additional nucleic acid molecules encoding polypeptidesthat confer desirable traits. The expression cassette will include inthe 5′-3′ direction of transcription, a transcriptional andtranslational initiation region (i.e., a promoter) and a polynucleotideopen reading frame. The expression cassette may optionally comprise atranscriptional and translational termination region (i.e. terminationregion) functional in plants. In some embodiments, the expressioncassette comprises a selectable marker gene to allow for selection forstable transformants. Expression constructs of the invention may alsocomprise a leader sequence and/or a sequence allowing for inducibleexpression of the polynucleotide of interest. See, Guo et al. (2003)Plant J. 34:383-92 and Chen et al. (2003) Plant J. 36:731-40 forexamples of sequences allowing for inducible expression.

The regulatory sequences of the expression construct are operably linkedto the polynucleotide of interest. By “operably linked” is intended afunctional linkage between a promoter and a second sequence wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleotide sequences being linked are contiguous.

Any promoter capable of driving expression in the plant of interest maybe used in the practice of the invention. The promoter may be native oranalogous or foreign or heterologous to the plant host. The terms“heterologous” and “exogenous” when used herein to refer to a nucleicacid sequence (e.g. a DNA or RNA sequence) or a gene, refer to asequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell but has been modified through,for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g.DNA or RNA) sequence naturally associated with a host cell into which itis introduced.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to that sequence. Thepromoters that are used for expression of the transgene(s) can be astrong plant promoter, a viral promoter, or a chimeric promoterscomposed of elements such as: TATA box from any gene (or synthetic,based on analysis of plant gene TATA boxes), optionally fused to theregion 5′ to the TATA box of plant promoters (which direct tissue andtemporally appropriate gene expression), optionally fused to 1 or moreenhancers (such as the 35S enhancer, FMV enhancer, CMP enhancer, RUBISCOSMALL SUBUNIT enhancer, PLASTOCYANIN enhancer).

Exemplary constitutive promoters include, for example, the core promoterof the Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Appropriate plant or chimeric promoters are useful for applications suchas expression of transgenes in certain tissues, while minimizingexpression in other tissues, such as seeds, or reproductive tissues.Exemplary cell type- or tissue-preferential promoters drive expressionpreferentially in the target tissue, but may also lead to someexpression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., PlantCell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988);Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., PlantPhysiology 110: 1069-1079 (1996).

In other embodiments of the present invention, inducible promoters maybe desired. Inducible promoters drive transcription in response toexternal stimuli such as chemical agents or environmental stimuli. Forexample, inducible promoters can confer transcription in response tohormones such as giberellic acid or ethylene, or in response to light ordrought.

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and correct mRNA polyadenylation. Thetermination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Appropriatetranscriptional terminators are those that are known to function inplants and include the CAMV 35S terminator, the tml terminator, thenopaline synthase terminator and the pea rbcs E9 terminator. These canbe used in both monocotyledons and dicotyledons. In addition, a gene'snative transcription terminator may be used.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues.

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adh1 gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1:1183-1200(1987)). In the same experimental system, the intron from the maizebronze 1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Samow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology84:965-968 (1987).

The present invention also relates to nucleic acid constructs comprisingone or more of the expression cassettes described above. The constructcan be a vector, such as a plant transformation vector. In oneembodiment, the vector is a plant transformation vector comprising apolynucleotide comprising the sequence set forth in SEQ ID NO:34, 35,36, or 37.

Plants

As used herein, the term “plant part” or “plant tissue” includes plantcells, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as embryos, pollen, ovules,seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, and the like.

Plants useful in the present invention include plants that aretransgenic for at least a polynucleotide encoding a mutant HPPDpolypeptide or variant thereof that retains HPPD enzymatic activity,alone or in combination with one or more additional nucleic acidmolecules encoding polypeptides that confer desirable traits. The typeof plant selected depends on a variety of factors, including forexample, the downstream use of the harvested plant material, amenabilityof the plant species to transformation, and the conditions under whichthe plants will be grown, harvested, and/or processed. One of skill willfurther recognize that additional factors for selecting appropriateplant varieties for use in the present invention include high yieldpotential, good stalk strength, resistance to specific diseases, droughttolerance, rapid dry down and grain quality sufficient to allow storageand shipment to market with minimum loss.

Plants according to the present invention include any plant that iscultivated for the purpose of producing plant material that is soughtafter by man or animal for either oral consumption, or for utilizationin an industrial, pharmaceutical, or commercial process. The inventionmay be applied to any of a variety of plants, including, but not limitedto maize, wheat, rice, barley, soybean, cotton, sorghum, beans ingeneral, rape/canola, alfalfa, flax, sunflower, safflower, millet, rye,sugarcane, sugar beet, cocoa, tea, Brassica, cotton, coffee, sweetpotato, flax, peanut, clover; vegetables such as lettuce, tomato,cucurbits, cassaya, potato, carrot, radish, pea, lentils, cabbage,cauliflower, broccoli, Brussels sprouts, peppers, and pineapple; treefruits such as citrus, apples, pears, peaches, apricots, walnuts,avocado, banana, and coconut; and flowers such as orchids, carnationsand roses. Other plants useful in the practice of the invention includeperennial grasses, such as switchgrass, prairie grasses, Indiangrass,Big bluestem grass and the like. It is recognized that mixtures ofplants may be used.

In addition, the term “crops” is to be understood as also includingcrops that have been rendered tolerant to herbicides or classes ofherbicides (such as, for example, ALS inhibitors, for exampleprimisulfuron, prosulfuron and trifloxysulfuron, EPSPS(5-enol-pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS(glutamine synthetase) inhibitors) as a result of conventional methodsof breeding or genetic engineering. Examples of crops that have beenrendered tolerant to herbicides or classes of herbicides by geneticengineering methods include glyphosate- and glufosinate-resistant cropvarieties commercially available under the trade names ROUNDUPREADY® andLIBERTYLINK®. The method according to the present invention isespecially suitable for the protection of soybean crops which have alsobeen rendered tolerant to glyphosate and/or glufosinate and where HPPDherbicides are used in a weed control programme along with other suchherbicides (glufosinate and/or glyphosate) for weed control.

It is further contemplated that the constructs of the invention may beintroduced into plant varieties having improved properties suitable oroptimal for a particular downstream use. For example,naturally-occurring genetic variability results in plants withresistance or tolerance to HPPD inhibitors or other herbicides, and suchplants are also useful in the methods of the invention. The methodaccording to the present invention can be further optimized by crossingthe transgenes that provide a level of tolerance, with soybean cultivarsthat exhibit an enhanced level of tolerance to HPPD inhibitors that isfound in a small percentage of soybean lines.

Plant Transformation

Once an herbicide resistant or tolerant mutant HPPD polynucleotide,alone or in combination with one or more additional nucleic acidmolecules encoding polypeptides that confer desirable traits, has beencloned into an expression system, it is transformed into a plant cell.The receptor and target expression cassettes of the present inventioncan be introduced into the plant cell in a number of art-recognizedways. The term “introducing” in the context of a polynucleotide, forexample, a nucleotide construct of interest, is intended to meanpresenting to the plant the polynucleotide in such a manner that thepolynucleotide gains access to the interior of a cell of the plant.Where more than one polynucleotide is to be introduced, thesepolynucleotides can be assembled as part of a single nucleotideconstruct, or as separate nucleotide constructs, and can be located onthe same or different transformation vectors. Accordingly, thesepolynucleotides can be introduced into the host cell of interest in asingle transformation event, in separate transformation events, or, forexample, in plants, as part of a breeding protocol. The methods of theinvention do not depend on a particular method for introducing one ormore polynucleotides into a plant, only that the polynucleotide(s) gainsaccess to the interior of at least one cell of the plant. Methods forintroducing polynucleotides into plants are known in the art including,but not limited to, transient transformation methods, stabletransformation methods, and virus-mediated methods.

“Transient transformation” in the context of a polynucleotide isintended to mean that a polynucleotide is introduced into the plant anddoes not integrate into the genome of the plant.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a plant is intended the introducedpolynucleotide is stably incorporated into the plant genome, and thusthe plant is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” is intended to mean thata polynucleotide, for example, a nucleotide construct described herein,introduced into a plant integrates into the genome of the plant and iscapable of being inherited by the progeny thereof, more particularly, bythe progeny of multiple successive generations.

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the pat and bar genes,which confer resistance to the herbicide glufosinate (also calledphosphinothricin; see White et al., Nucl. Acids Res 18: 1062 (1990),Spencer et al. Theor. Appl. Genet 79: 625-631 (1990) and U.S. Pat. Nos.5,561,236 and 5,276,268), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell. Biol. 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642), the glyphosate N-acetyltransferase (GAT) gene, which alsoconfers resistance to glyphosate (Castle et al. (2004) Science,304:1151-1154; U.S. Patent App. Pub. Nos. 20070004912, 20050246798, and20050060767); and the mannose-6-phosphate isomerase gene, which providesthe ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and5,994,629). Alternatively, and in one preferred embodiment the HPPD geneof the current invention is, in combination with the use of an HPPDherbicide as selection agent, itself used as the selectable marker.

Methods for regeneration of plants are also well known in the art. Forexample, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,microinjection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of representative techniques for transforming bothdicotyledonous and monocotyledonous plants, as well as a representativeplastid transformation technique.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Forthe construction of vectors useful in Agrobacterium transformation, see,for example, US Patent Application Publication No. 2006/0260011, hereinincorporated by reference. Transformation without the use ofAgrobacterium tumefaciens circumvents the requirement for T-DNAsequences in the chosen transformation vector and consequently vectorslacking these sequences can be utilized in addition to vectors such asthe ones described above which contain T-DNA sequences. Transformationtechniques that do not rely on Agrobacterium include transformation viaparticle bombardment, protoplast uptake (e.g. PEG and electroporation)and microinjection. The choice of vector depends largely on thepreferred selection for the species being transformed. For theconstruction of such vectors, see, for example, US Application No.20060260011, herein incorporated by reference.

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011, SeeExample 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J. 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth of these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5-2.5 mm length excised from a maize ear 14-15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8:736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation,transformation and regeneration of Pooideae protoplasts. Thesetechniques allow the transformation of Dactylis and wheat. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10: 667-674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102:1077-1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus. A preferred technique for wheat transformation,however, involves the transformation of wheat by particle bombardment ofimmature embryos and includes either a high sucrose or a high maltosestep prior to gene delivery. Prior to bombardment, any number of embryos(0.75-1 mm in length) are plated onto MS medium with 3% sucrose(Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l2,4-D for induction of somatic embryos, which is allowed to proceed inthe dark. On the chosen day of bombardment, embryos are removed from theinduction medium and placed onto the osmoticum (i.e. induction mediumwith sucrose or maltose added at the desired concentration, typically15%). The embryos are allowed to plasmolyze for 2-3 hours and are thenbombarded. Twenty embryos per target plate is typical, although notcritical. An appropriate gene-carrying plasmid (such as pCIB3064 orpSOG35) is precipitated onto micrometer size gold particles usingstandard procedures. Each plate of embryos is shot with the DuPontBIOLISTICS® helium device using a burst pressure of about 1000 psi usinga standard 80 mesh screen. After bombardment, the embryos are placedback into the dark to recover for about 24 hours (still on osmoticum).After 24 hrs, the embryos are removed from the osmoticum and placed backonto induction medium where they stay for about a month beforeregeneration. Approximately one month later the embryo explants withdeveloping embryogenic callus are transferred to regeneration medium(MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriateselection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/lmethotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contain half-strength MS, 2% sucrose, and the sameconcentration of selection agent.

Transformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of whichare incorporated herein by reference. See also, Negrotto et al., PlantCell Reports 19: 798-803 (2000), incorporated herein by reference.

For example, rice (Oryza sativa) can be used for generating transgenicplants. Various rice cultivars can be used (Hiei et al., 1994, PlantJournal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276; Hieiet al., 1997, Plant Molecular Biology, 35:205-218). Also, the variousmedia constituents described below may be either varied in quantity orsubstituted. Embryogenic responses are initiated and/or cultures areestablished from mature embryos by culturing on MS-CIM medium (MS basalsalts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 gaiter;proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH;Phytagel, 3 g/liter). Either mature embryos at the initial stages ofculture response or established culture lines are inoculated andco-cultivated with the Agrobacterium tumefaciens strain LBA4404(Agrobacterium) containing the desired vector construction.Agrobacterium is cultured from glycerol stocks on solid YPC medium (100mg/L spectinomycin and any other appropriate antibiotic) for about 2days at 28° C. Agrobacterium is re-suspended in liquid MS-CIM medium.The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 andacetosyringone is added to a final concentration of 200 uM.Acetosyringone is added before mixing the solution with the ricecultures to induce Agrobacterium for DNA transfer to the plant cells.For inoculation, the plant cultures are immersed in the bacterialsuspension. The liquid bacterial suspension is removed and theinoculated cultures are placed on co-cultivation medium and incubated at22° C. for two days. The cultures are then transferred to MS-CIM mediumwith Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium.For constructs utilizing the PMI selectable marker gene (Reed et al., InVitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred toselection medium containing Mannose as a carbohydrate source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4weeks in the dark. Resistant colonies are then transferred toregeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) andgrown in the dark for 14 days. Proliferating colonies are thentransferred to another round of regeneration induction media and movedto the light growth room. Regenerated shoots are transferred to GA7containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2weeks and then moved to the greenhouse when they are large enough andhave adequate roots. Plants are transplanted to soil in the greenhouse(To generation) grown to maturity, and the T₁ seed is harvested.

The plants obtained via transformation with a nucleic acid sequence ofinterest in the present invention can be any of a wide variety of plantspecies, including those of monocots and dicots; however, the plantsused in the method of the invention are preferably selected from thelist of agronomically important target crops set forth elsewhere herein.The expression of a gene of the present invention in combination withother characteristics important for production and quality can beincorporated into plant lines through breeding. Breeding approaches andtechniques are known in the art. See, for example, Welsh J. R.,Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY(1981); Crop Breeding, Wood D. R. (Ed.) American Society of AgronomyMadison, Wis. (1983); Mayo O., The Theory of Plant Breeding, SecondEdition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding forResistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); andWricke and Weber, Quantitative Genetics and Selection Plant Breeding,Walter de Gruyter and Co., Berlin (1986).

For the transformation of plastids, seeds of Nicotiana tabacum c.v.“Xanthienc” are germinated seven per plate in a 1″ circular array on Tagar medium and bombarded 12-14 days after sowing with 1 um tungstenparticles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmidspPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P.(1993) PNAS 90, 913-917). Bombarded seedlings are incubated on T mediumfor two days after which leaves are excised and placed abaxial side upin bright light (350-500 umol photons/m²/s) on plates of RMOP medium(Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530)containing 500 ug/ml spectinomycin dihydrochloride (Sigma, St. Louis,Mo.). Resistant shoots appearing underneath the bleached leaves three toeight weeks after bombardment are subcloned onto the same selectivemedium, allowed to form callus, and secondary shoots isolated andsubcloned. Complete segregation of transformed plastid genome copies(homoplasmicity) in independent subclones is assessed by standardtechniques of Southern blotting (Sambrook et al., (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987)Plant Mol Biol Reporter 5, 346349) is separated on 1% Tris-borate (TBE)agarose gels, transferred to nylon membranes (Amersham) and probed with.sup.32P-labeled random primed DNA sequences corresponding to a 0.7 kbBamHI/HindIII DNA fragment from pC8 containing a portion of the rps7/12plastid targeting sequence. Homoplasmic shoots are rootedaseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. etal. (1994) PNAS 91, 7301-7305) and transferred to the greenhouse.

The genetic properties engineered into the transgenic seeds and plantsdescribed above are passed on by sexual reproduction or vegetativegrowth and can thus be maintained and propagated in progeny plants.Generally, maintenance and propagation make use of known agriculturalmethods developed to fit specific purposes such as tilling, sowing orharvesting.

Use of the advantageous genetic properties of the transgenic plants andseeds according to the invention can further be made in plant breeding.Depending on the desired properties, different breeding measures aretaken. The relevant techniques are well known in the art and include butare not limited to hybridization, inbreeding, backcross breeding,multi-line breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Thus, the transgenic seeds and plantsaccording to the invention can be used for the breeding of improvedplant lines that, for example, increase the effectiveness ofconventional methods such as herbicide or pesticide treatment or allowone to dispense with said methods due to their modified geneticproperties.

Many suitable methods for transformation using suitable selectionmarkers such as kanamycin, binary vectors such as from Agrobacterium andplant regeneration as, for example, from tobacco leaf discs are wellknown in the art. Optionally, a control population of plants arelikewise transformed with a polynucleotide expressing the control HPPD.Alternatively, an untransformed dicot plant such as Arabidopsis orTobacco can be used as a control since this, in any case, expresses itsown endogenous HPPD.

Herbicide Resistance

The present invention provides transgenic plants, plant cells, tissues,and seeds that have been transformed with a nucleic acid moleculeencoding a mutant HPPD or variant thereof that confers resistance ortolerance to herbicides, alone or in combination with one or moreadditional nucleic acid molecules encoding polypeptides that conferdesirable traits.

In one embodiment, the transgenic plants of the invention exhibitresistance or tolerance to application of herbicide in an amount of fromabout 5 to about 2,000 grams per hectare (g/ha), including, for example,about 5 g/ha, about 10 g/ha, about 15 g/ha, about 20 g/ha, about 25g/ha, about 30 g/ha, about 35 g/ha, about 40 g/ha, about 45 g/ha, about50 g/ha, about 55 g/ha, about 60 g/ha, about 65 g/ha, about 70 g/ha,about 75 g/ha, about 80 g/ha, about 85 g/ha, about 90 g/ha, about 95g/ha, about 100 g/ha, about 110 g/ha, about 120 g/ha, about 130 g/ha,about 140 g/ha, about 150 g/ha, about 160 g/ha, about 170 g/ha, about180 g/ha, about 190 g/ha, about 200 g/ha, about 210 g/ha, about 220g/ha, about 230 g/ha, about 240 g/ha, about 250 g/ha, about 260 g/ha,about 270 g/ha, about 280 g/ha, about 290 g/ha, about 300 g/ha, about310 g/ha, about 320 g/ha, about 330 g/ha, about 340 g/ha, about 350g/ha, about 360 g/ha, about 370 g/ha, about 380 g/ha, about 390 g/ha,about 400 g/ha, about 410 g/ha, about 420 g/ha, about 430 g/ha, about440 g/ha, about 450 g/ha, about 460 g/ha, about 470 g/ha, about 480g/ha, about 490 g/ha, about 500 g/ha, about 510 g/ha, about 520 g/ha,about 530 g/ha, about 540 g/ha, about 550 g/ha, about 560 g/ha, about570 g/ha, about 580 g/ha, about 590 g/ha, about 600 g/ha, about 610g/ha, about 620 g/ha, about 630 g/ha, about 640 g/ha, about 650 g/ha,about 660 g/ha, about 670 g/ha, about 680 g/ha, about 690 g/ha, about700 g/ha, about 710 g/ha, about 720 g/ha, about 730 g/ha, about 740g/ha, about 750 g/ha, about 760 g/ha, about 770 g/ha, about 780 g/ha,about 790 g/ha, about 800 g/ha, about 810 g/ha, about 820 g/ha, about830 g/ha, about 840 g/ha, about 850 g/ha, about 860 g/ha, about 870g/ha, about 880 g/ha, about 890 g/ha, about 900 g/ha, about 910 g/ha,about 920 g/ha, about 930 g/ha, about 940 g/ha, about 950 g/ha, about960 g/ha, about 970 g/ha, about 980 g/ha, about 990 g/ha, about 1,000,g/ha, about 1,010 g/ha, about 1,020 g/ha, about 1,030 g/ha, about 1,040g/ha, about 1,050 g/ha, about 1,060 g/ha, about 1,070 g/ha, about 1,080g/ha, about 1,090 g/ha, about 1,100 g/ha, about 1,110 g/ha, about 1,120g/ha, about 1,130 g/ha, about 1,140 g/ha, about 1,150 g/ha, about 1,160g/ha, about 1,170 g/ha, about 1,180 g/ha, about 1,190 g/ha, about 1,200g/ha, about 1,210 g/ha, about 1,220 g/ha, about 1,230 g/ha, about 1,240g/ha, about 1,250 g/ha, about 1,260 g/ha, about 1,270 g/ha, about 1,280g/ha, about 1,290 g/ha, about 1,300 g/ha, about 1,310 g/ha, about 1,320g/ha, about 1,330 g/ha, about 1,340 g/ha, about 1,350 g/ha, about 360g/ha, about 1,370 g/ha, about 1,380 g/ha, about 1,390 g/ha, about 1,400g/ha, about 1,410 g/ha, about 1,420 g/ha, about 1,430 g/ha, about 1,440g/ha, about 1,450 g/ha, about 1,460 g/ha, about 1,470 g/ha, about 1,480g/ha, about 1,490 g/ha, about 1,500 g/ha, about 1,510 g/ha, about 1,520g/ha, about 1,530 g/ha, about 1,540 g/ha, about 1,550 g/ha, about 1,560g/ha, about 1,570 g/ha, about 1,580 g/ha, about 1,590 g/ha, about 1,600g/ha, about 1,610 g/ha, about 1,620 g/ha, about 1,630 g/ha, about 1,640g/ha, about 1,650 g/ha, about 1,660 g/ha, about 1,670 g/ha, about 1,680g/ha, about 1,690 g/ha, about 1,700 g/ha, about 1,710 g/ha, about 1,720g/ha, about 1,730 g/ha, about 1,740 g/ha, about 1,750 g/ha, about 1,760g/ha, about 1,770 g/ha, about 1,780 g/ha, about 1,790 g/ha, about 1,800g/ha, about 1,810 g/ha, about 1,820 g/ha, about 1,830 g/ha, about 1,840g/ha, about 1,850 g/ha, about 1,860 g/ha, about 1,870 g/ha, about 1,880g/ha, about 1,890 g/ha, about 1,900 g/ha, about 1,910 g/ha, about 1,920g/ha, about 1,930 g/ha, about 1,940 g/ha, about 1,950 g/ha, about 1,960g/ha, about 1,970 g/ha, about 1,980 g/ha, about 1,990 g/ha, or about2,000.

The average and distribution of herbicide tolerance or resistance levelsof a range of primary plant transformation events are evaluated in thenormal manner based upon plant damage, meristematic bleaching symptomsetc. at a range of different concentrations of herbicides. These datacan be expressed in terms of, for example, GR50 values derived fromdose/response curves having “dose” plotted on the x-axis and “percentagekill”, “herbicidal effect”, “numbers of emerging green plants” etc.plotted on the y-axis where increased GR50 values correspond toincreased levels of inherent inhibitor-tolerance (e.g. increasedKi/Km_(HPP) value) and/or level of expression of the expressed HPPDpolypeptide.

The methods of the present invention are especially useful to protectcrops from the herbicidal injury of HPPD inhibitor herbicides of theclasses of HPPD chemistry described below. In one embodiment, theselected from the group consisting of:

a) a compound of formula (Ia)

wherein R¹ and R² are hydrogen or together form an ethylene bridge;R³ is hydroxy or phenylthio-; R⁴ is halogen, nitro, C₁-C₄alkyl,C₁-C₄alkoxy-C₁-C₄alkyl-, C₁-C₄alkoxy-C₁-C₄alkoxy-C₁-C₄alkyl-;X is methine, nitrogen, or C—R⁵ wherein R⁵ is hydrogen, C₁-C₄alkoxy,C₁-C₄haloalkoxy-C₁-C₄alkyl-, or a group

andR⁶ is C₁-C₄alkylsulfonyl- or C₁-C₄haloalkyl;

b) a compound of formula (Ib)

R¹ and R² are independently C₁-C₄alkyl; and the free acids thereof;

c) a compound of formula (Ic)

wherein R¹ is hydroxy, phenylcarbonyl-C₁-C₄alkoxy- orphenylcarbonyl-C₁-C₄alkoxy- wherein the phenyl moiety is substituted inpara-position by halogen or C₁-C₄alkyl, or phenylsulfonyloxy- orphenylsulfonyloxy- wherein the phenyl moiety is substituted inpara-position by halogen or C₁-C₄alkyl;R² is C₁-C₄alkyl;R³ is hydrogen or C₁-C₄alkyl; R⁴ and R⁶ are independently halogen,C₁-C₄alkyl, C₁-C₄haloalkyl, or C₁-C₄alkylsulfonyl-; andR⁵ is hydrogen, C₁-C₄alkyl, C₁-C₄alkoxy-C₁-C₄alkoxy-, or a group

d) a compound of formula (Id)

wherein R¹ is hydroxy;R² is C₁-C₄alkyl;R³ is hydrogen; and R⁴, R⁵ and R⁶ are independently C₁-C₄alkyl;

e) a compound of formula (Ie)

wherein R¹ is cyclopropyl;R² and R⁴ are independently halogen, C₁-C₄haloalkyl, orC₁-C₄alkylsulfonyl-; andR³ is hydrogen;

f) a compound of formula (If)

wherein R¹ is cyclopropyl;R² and R⁴ are independently halogen, C₁-C₄haloalkyl, orC₁-C₄alkylsulfonyl-; andR³ is hydrogen;

g) a compound of formula (Ig) or Formula (Ih)

wherein:R² is selected from the group consisting of C₁-C₃alkyl, C₁-C₃haloalkyl,C₁-C₃alkoxy-C₁-C₃ alkyl and C₁-C₃ alkoxy-C₂-C₃alkoxy-C₁-C₃-alkyl;R⁵ is hydrogen or methyl;R⁶ is selected from the group consisting of hydrogen, fluorine,chlorine, hydroxyl and methyl;R⁷ is selected from the group consisting of hydrogen, halogen, hydroxyl,sulfhydryl, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆haloalkyl,C₂-C₆haloalkenyl, C₂-C₆alkenyl, C₃-C₆alkynyl, C₁-C₆alkoxy,C₄-C₇cycloalkoxy, C₁-C₆haloalkoxy, C₁-C₆alkylthio, C₁-C₆alkylsulfinyl,C₁-C₆alkylsulfonyl, C₁-C₆haloalkylthio, amino, C₁-C₆alkylamino,C₂-C₆dialkylamino, C₂-C₆dialkylaminosulfonyl, C₁-C₆alkylaminosulfonyl,C₁-C₆alkoxy-C₁-C₆alkyl, C₁-C₆alkoxy-C₂-C₆alkoxy, C₁-C₆alkoxy-C₂-C₆alkoxy-C₁-C₆-alkyl, C₃-C₆alkenyl-C₂-C₆alkoxy, C₃-C₆alkynyl-C₁-C₆alkoxy,C₁-C₆alkoxycarbonyl, C₁-C₆alkylcarbonyl, C₁-C₄alkylenyl-S(O)p-R′,C₁-C₄alkylenyl-CO₂—R′, C₁-C₄alkylenyl-(CO)N—R′R′, phenyl, phenylthio,phenylsulfinyl, phenylsulfonyl, phenoxy, pyrrolidinyl, piperidinyl,morpholinyl and 5 or 6-membered heteroaryl or heteroaryloxy, theheteroaryl containing one to three heteroatoms, each independentlyselected from the group consisting of oxygen, nitrogen and sulphur,wherein the phenyl or heteroaryl component may be optionally substitutedby a substituent selected from the group consisting of C₁-C₃alkyl,C₁-C₃haloalkyl, C₁-C₃ alkoxy, C₁-C₃haloalkoxy, halo, cyano, and nitro;

X=O or S;

n=0 or 1;m=0 or 1 with the proviso that if m=1 then n=0 and if n=1 then m=0;p=0, 1, or 2;R′ is independently selected from the group consisting of hydrogen andC₁-C₆alkyl;R⁸ is selected from the group consisting of hydrogen, C₁-C₆alkyl,C₁-C₆haloalkyl, C₁-C₆alkylcarbonyl-C₁-C₃alkyl, C₃-C₆cycloalkylalkeneylfor example cyclohexylmethylenyl, C₃-C₆alkynylalkyleneyl for examplepropargyl, C₂-C₆-alkenylalkylenyl for example allyl, C₁-C₆alkoxyC₁-C₆alkyl, cyano-C₁-C₆-alkyl, arylcarbonyl-C₁-C₃-alkyl (wherein thearyl may be optionally substituted with a substituent selected from thegroup consisting of halo, C₁-C₃-alkoxy, C₁-C₃-alkyl, C₁-C₃ haloalkyl),aryl-C₁-C₆alkyl (wherein the aryl may be optionally substituted with asubstituent selected from the group consisting of halo, C₁-C₃-alkoxy,C₁-C₃-alkyl, C₁-C₃ haloalkyl), C₁-C₆alkoxy C₁-C₆alkoxy C₁-C₆alkyl and a5 or 6-membered heteroaryl-C₁-C₃-alkyl or heterocyclyl-C₁-C₃-alkyl, theheteroaryl or heterocyclyl containing one to three heteroatoms, eachindependently selected from the group consisting of oxygen, nitrogen andsulphur, wherein the heterocyclyl or heteroaryl component may beoptionally substituted by a substituent selected from the groupconsisting of halo, C₁-C₃alkyl, C₁-C₃haloalkyl, and C₁-C₃ alkoxy;Q is selected from the group consisting of:

whereinA¹ is selected from the group consisting of O, C(O), S, SO, SO₂ and(CR^(e)R^(f))_(q);q=0, 1 or 2;R^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) are each independentlyselected from the group consisting of C₁-C₄alkyl which may be mono-, di-or tri-substituted by substituents selected from the group consisting ofC₁-C₄alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylthio, C₁-C₄alkylsulfinyl,C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl, phenyl and heteroaryl, it beingpossible for the phenyl and heteroaryl groups in turn to be mono-, di-or tri-substituted by substituents selected from the group consisting ofC₁-C₄alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl and C₁-C₄haloalkyl, thesubstituents on the nitrogen in the heterocyclic ring being other thanhalogen; orR^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) are each independentlyselected from the group consisting of hydrogen, C₁-C₄alkoxy, halogen,hydroxy, cyano, hydroxycarbonyl, C₁-C₄alkoxycarbonyl, C₁-C₄alkylthio,C₁-C₄alkylsulfinyl, C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl, phenyl orheteroaryl, it being possible for the phenyl and heteroaryl groups inturn to be mono-, di- or tri-substituted by substituents selected fromthe group consisting of C₁-C₄alkoxy, halogen, hydroxy, cyano,hydroxycarbonyl, C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl andC₁-C₄haloalkyl, the substituents on the nitrogen in the heterocyclicring being other than halogen; orR^(a) and R^(b) together form a 3- to 5-membered carbocyclic ring whichmay be substituted by C₁-C₄alkyl and may be interrupted by oxygen,sulfur, S(O), SO₂, OC(O), NR^(g) or by C(O); orR^(a) and R^(c) together form a C₁-C₃alkylene chain which may beinterrupted by oxygen, sulfur, SO, SO₂, OC(O), NR^(h) or by C(O); itbeing possible for that C₁-C₃alkylene chain in turn to be substituted byC₁-C₄alkyl;R^(g) and R^(h) are each independently of the other C₁-C₄alkyl,C₁-C₄haloalkyl, C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl orC₁-C₄alkoxycarbonyl;R^(i) is C₁-C₄alkyl;R³ is selected from the group consisting of C₁-C₆alkyl, optionallysubstituted with halogen and/or C₁-C₃alkoxy; and C₃-C₆ cycloalkyloptionally substituted with halogen and/or C₁-C₃alkoxy;R⁹ is selected from the group consisting of cyclopropyl, CF₃ and i.-Pr;R¹⁰ is selected from the group consisting of hydrogen, I, Br, SR¹¹,S(O)R¹¹, S(O)₂R¹¹ and CO₂R¹¹; andR¹¹ is C₁₋₄ alkyl;h) a compound of formula (Ij), (Ik), or (Im)

or an agronomically acceptable salt of said compound, wherein:R¹ is selected from the group consisting of hydrogen, C₁-C₆alkyl,C₁-C₆haloalkyl, C₁-C₃alkoxy-C₁-C₃ alkyl, C₁-C₃alkoxy-C₁-C₃alkoxy-C₁-C₃-alkyl, C₁-C₃alkoxy-C₁-C₃-haloalkyl,C₁-C₃-alkoxy-C₁-C₃-alkoxy-C₁-C₃-haloalkyl, C₄-C₆-oxasubstitutedcycloalkoxy-C₁-C₃-alkyl, C₄-C₆-oxasubstitutedcycloalkyl-C₁-C₃-alkoxy-C₁-C₃-alkyl, C₄-C₆-oxasubstitutedcycloalkoxy-C₁-C₃-haloalkyl, C₄-C₆-oxasubstitutedcycloalkyl-C₁-C₃-alkoxy-C₁-C₃-halo alkyl, (C₁-C₃ alkanesulfonyl-C₁-C₃alkylamino)-C₁-C₃ alkyl, (C₁-C₃ alkanesulfonyl-C₃-C₄cycloalkylamino)-C₁-C₃alkyl, C₁-C₆alkylcarbonyl-C₁-C₃alkyl,C₃-C₆cycloalkyl-C₂-C₆alkenyl, C₃-C₆alkynyl, C₂-C₆-alkenyl,cyano-C₁-C₆-alkyl, arylcarbonyl-C₁-C₃-alkyl (wherein the aryl may beoptionally substituted with one or more substituents from the groupconsisting of halo, C₁-C₃-alkoxy, C₁-C₃-alkyl, C₁-C₃ haloalkyl),aryl-C₁-C₆alkyl (wherein the aryl may be optionally substituted with oneor more substituents from the group consisting of halo, C₁-C₃-alkoxy,C₁-C₃-alkyl, C₁-C₃ haloalkyl), aryl, 5 or 6-membered heteroaryl, 5 or6-membered heteroaryl-C₁-C₃alkyl and heterocyclyl-C₁-C₃alkyl, theheteroaryl or heterocyclyl containing one to three heteroatoms eachindependently selected from the group consisting of oxygen, nitrogen andsulphur, and wherein the aryl, heterocyclyl or heteroaryl component maybe optionally substituted by one or more substituents selected from thegroup consisting of halo, C₁-C₃alkyl, C₁-C₃haloalkyl, C₁-C₃ alkoxy,C₁-C₃ haloalkoxy, C₁-C₆alkyl-S(O)p-, C₁-C₆haloalkyl-S(O)p-, cyano andnitro;R⁵ is selected from the group consisting of hydrogen, chloro, fluoro andmethyl;R⁶ is selected from the group consisting of hydrogen, fluorine,chlorine, hydroxyl and methyl;R⁷ is selected from the group consisting of hydrogen, cyano, nitro,halogen, hydroxyl, sulfhydryl, C₁-C₆alkyl, C₃-C₆cycloalkyl,C₁-C₆haloalkyl, C₂-C₆haloalkenyl, C₂-C₆alkenyl, aryl-C₂-C₆alkenyl,C₃-C₆alkynyl, C₁-C₆alkoxy, C₄-C₇ cycloalkoxy, C₁-C₆haloalkoxy,C₁-C₆alkyl-S(O)p, C₃-C₆cycloalkyl-S(O)pC₁-C₆haloalkyl-S(O)_(p), C₃-C₆halocycloalkyl-S(O)p, C₁-C₆alkylcarbonylamino,(C₁-C₆alkylcarbonyl)C₁-C₃alkylamino, (C₃-C₆cycloalkylcarbonyl)amino,(C₃-C₆cycloalkylcarbonyl)C₁-C₃alkylamino, arylcarbonylamino,(arylcarbonyl)-C₁₋₃alkylamino, (heteroarylcarbonyl)amino,(heteroarylcarbonyl)C₁-C₃alkylamino, amino, C₁-C₆alkylamino,C₂-C₆dialkylamino, C₂-C₆alkenylamino, C₁-C₆alkoxy-C₂-C₆-alkylamino,(C₁-C₆alkoxy-C₂-C₄-alkyl)-C₁-C₆-alkylamino, C₃-C₆ cycloalkylamino, C₃-C₆cyclohaloalkylamino, C₁-C₃alkoxy-C₃-C₆ cycloalkylamino, C₃-C₆alkynylamino, dialkylamino in which the substituents join to form a 4-6membered ring (e.g. pyrrolidinyl, piperidinyl) optionally containingoxygen (e.g. morpholinyl) and/or optionally substituted by C₁-C₃-alkoxyand/or halogen (especially fluorine), C₂-C₆dialkylaminosulfonyl,C₁-C₆alkylaminosulfonyl, C₁-C₆alkoxy-C₁-C₆alkyl,C₁-C₆alkoxy-C₂-C₆alkoxy, C₁-C₆alkoxy-C₂-C₆ alkoxy-C₁-C₆-alkyl,C₃-C₆alkenyl-C₂-C₆alkoxy, C₃-C₆alkynyl-C₁-C₆alkoxy, C₁-C₆alkoxycarbonyl,C₁-C₆alkylcarbonyl, C₁-C₄alkylenyl-S(O)p—R′, C₁-C₄alkylenyl-CO₂—R′,C₁-C₄alkylenyl-(CO)N—R′R′, aryl (e.g. phenyl), aryl C₁-C₃alkyl,aryl-S(O)p, heteroaryl-S(O)p, aryloxy (e.g. phenoxy), a 5 or 6-memberedheteroaryl, heteroaryl C₁-C₃ alkyl and heteroaryloxy, the heteroarylcontaining one to three heteroatoms, each independently selected fromthe group consisting of oxygen, nitrogen and sulphur, wherein the arylor heteroaryl component may be optionally substituted by one or moresubstituents selected from the group consisting of C₁-C₃alkyl,C₁-C₃haloalkyl, C₁-C₃ alkoxy, C₁-C₃haloalkoxy, halo, cyano and nitro;

X¹=N—(O)n or C—R⁸; X²=O or S;

n=0 or 1;p=0, 1 or 2;R′ is independently selected from the group consisting of hydrogen andC₁-C₆alkyl;R⁸ is selected from the group consisting of hydrogen, halogen,C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkylcarbonyl-C₁-C₃alkyl,C₃-C₆cycloalkyl-C₂-C₆alkenyl for example cyclohexylmethylenyl,C₃-C₆alkynyl (for example propargyl), C₂-C₆-alkenyl (for example allyl),C₁-C₆alkoxy C₁-C₆alkyl, cyano-C₁-C₆-alkyl, arylcarbonyl-C₁-C₃-alkyl(wherein the aryl may be optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₃-alkoxy,C₁-C₃-alkyl, C₁-C₃ haloalkyl), aryl-C₁-C₆alkyl (wherein the aryl may beoptionally substituted with one or more substituents from the groupconsisting of halo, C₁-C₃-alkoxy, C₁-C₃-alkyl, C₁-C₃ haloalkyl),C₁-C₆alkoxyC₁-C₆alkoxy C₁-C₆alkyl, aryl, a 5 or 6-membered heteroaryl, a5 or 6-membered heteroaryl-C₁-C₃-alkyl and heterocyclyl-C₁-C₃-alkyl, theheteroaryl or heterocyclyl containing one to three heteroatoms eachindependently selected from the group consisting of oxygen, nitrogen andsulphur, and wherein the aryl, heterocyclyl or heteroaryl component maybe optionally substituted by one or more substituents from the groupconsisting of halogen, C₁-C₃alkyl, C₁-C₃haloalkyl and C₁-C₃ alkoxy,cyano and nitro;Q is selected from the group consisting of: —

whereinA¹ is selected from the group consisting of O, C(O), S, SO, SO₂ and(CR^(e)R^(f))_(q);q=0, 1 or 2;R^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) are each independentlyselected from the group consisting of C₁-C₄alkyl which may be mono-, di-or tri-substituted by substituents selected from the group consisting ofC₁-C₄alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylthio, C₁-C₄alkylsulfinyl,C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl, phenyl and heteroaryl, it beingpossible for the phenyl and heteroaryl groups in turn to be mono-, di-or tri-substituted by substituents selected from the group consisting ofC₁-C₄alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl and C₁-C₄haloalkyl, thesubstituents on the nitrogen in the heterocyclic ring being other thanhalogen; orR^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) are each independentlyselected from the group consisting of hydrogen, C₁-C₄alkoxy, halogen,hydroxy, cyano, hydroxycarbonyl, C₁-C₄alkoxycarbonyl, C₁-C₄alkylthio,C₁-C₄alkylsulfinyl, C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl, phenyl orheteroaryl, it being possible for the phenyl and heteroaryl groups inturn to be mono-, di- or tri-substituted by substituents selected fromthe group consisting of C₁-C₄alkoxy, halogen, hydroxy, cyano,hydroxycarbonyl, C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl andC₁-C₄haloalkyl, the substituents on the nitrogen in the heterocyclicring being other than halogen; orR^(a) and R^(b) together form a 3- to 5-membered carbocyclic ring whichmay be substituted by C₁-C₄alkyl and may be interrupted by oxygen,sulfur, S(O), SO₂, OC(O), NR⁹ or by C(O); orR^(a) and R^(e) together form a C₁-C₃alkylene chain which may beinterrupted by oxygen, sulfur, SO, SO₂, OC(O), NR^(h) or by C(O); itbeing possible for that C₁-C₃alkylene chain in turn to be substituted byC₁-C₄alkyl;R^(g) and R^(h) are each independently of the other C₁-C₄alkyl,C₁-C₄haloalkyl, C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl orC₁-C₄alkoxycarbonyl;R^(i) is C₁-C₄alkyl;R^(j) is selected from the group consisting of hydrogen, C₁-C₄ alkyl andC₃-C₆ cycloalkyl;R³ is selected from the group consisting of C₁-C₆alkyl, optionallysubstituted with halogen and/or C₁-C₃alkoxy, and C₃-C₆ cycloalkyloptionally substituted with halogen and/or C₁-C₃alkoxy;R⁹ is selected from the group consisting of cyclopropyl, CF₃ and i.-Pr;R¹⁰ is selected from the group consisting of hydrogen, I, Br, SR¹¹,S(O)R¹¹, S(O)₂R¹¹ and CO₂R¹¹; andR¹¹ is C₁₋₄ alkyl.

With respect to the structures (Ia)-(Im) described herein:

Halogen encompasses fluorine, chlorine, bromine or iodine. The samecorrespondingly applies to halogen in the context of other definitions,such as haloalkyl or halophenyl.

Haloalkyl groups having a chain length of from 1 to 6 carbon atoms are,for example, fluoromethyl, difluoromethyl, trifluoromethyl,chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl,2-fluoroethyl, 2-chloroethyl, pentafluoroethyl,1,1-difluoro-2,2,2-trichloroethyl, 2,2,3,3-tetrafluoroethyl and2,2,2-trichloroethyl, heptafluoro-n-propyl and perfluoro-n-hexyl.

Suitable alkylenyl radicals include, for example CH₂, CHCH₃, C(CH₃)₂,CH₂CHCH₃, CH₂CH(C₂H₅).

Suitable haloalkenyl radicals include alkenyl groups substituted one ormore times by halogen, halogen being fluorine, chlorine, bromine oriodine and especially fluorine or chlorine, for example2,2-difluoro-1-methylvinyl, 3-fluoropropenyl, 3-chloropropenyl,3-bromopropenyl, 2,3,3-trifluoropropenyl, 2,3,3-trichloropropenyl and4,4,4-trifluorobut-2-en-1-yl. Preferred C₂-C₆alkenyl radicalssubstituted once, twice or three times by halogen are those having achain length of from 2 to 5 carbon atoms. Suitable haloalkylalkynylradicals include, for example, alkylalkynyl groups substituted one ormore times by halogen, halogen being bromine or iodine and, especially,fluorine or chlorine, for example 3-fluoropropynyl,5-chloropent-2-yn-1-yl, 5-bromopent-2-yn-1-yl, 3,3,3-trifluoropropynyland 4,4,4-trifluoro-but-2-yn-1-yl. Preferred alkylalkynyl groupssubstituted one or more times by halogen are those having a chain lengthof from 3 to 5 carbon atoms.

Alkoxy groups preferably have a chain length of from 1 to 6 carbonatoms. Alkoxy is, for example, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, isobutoxy, sec-butoxy or tert-butoxy or a pentyloxy orhexyloxy isomer, preferably methoxy and ethoxy. Alkylcarbonyl ispreferably acetyl or propionyl. Alkoxycarbonyl is, for example,methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,n-butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl ortert-butoxycarbonyl, preferably methoxycarbonyl, ethoxycarbonyl ortert-butoxycarbonyl.

Haloalkoxy is, for example, fluoromethoxy, difluoromethoxy,trifluoromethoxy, 2,2,2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy,2-fluoroethoxy, 2-chloroethoxy, 2,2-difluoroethoxy or2,2,2-trichloroethoxy, preferably difluoromethoxy, 2-chloroethoxy ortrifluoromethoxy.

Alkylthio groups preferably have a chain length of from 1 to 6 carbonatoms. Alkylthio is, for example, methylthio, ethylthio, propylthio,isopropylthio, n-butylthio, isobutylthio, sec-butylthio ortert-butylthio, preferably methylthio or ethylthio. Alkylsulfinyl is,for example, methylsulfinyl, ethylsulfinyl, propylsulfinyl,isopropylsulfinyl, n-butylsulfinyl, isobutylsulfinyl, sec-butylsulfinylor tert-butylsulfinyl, preferably methylsulfinyl or ethylsulfinyl.

Alkylsulfonyl is, for example, methylsulfonyl, ethylsulfonyl,propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, isobutylsulfonyl,sec-butylsulfonyl or tert-butylsulfonyl, preferably methylsulfonyl orethylsulfonyl.

Alkylamino is, for example, methylamino, ethylamino, n-propylamino,isopropylamino or a butylamino isomer. Dialkylamino is, for example,dimethylamino, methylethylamino, diethylamino, n-propylmethylamino,dibutylamino or diisopropylamino. Preference is given to alkylaminogroups having a chain length of from 1 to 4 carbon atoms.

Cycloalkylamino or dicycloalkylamino is for example cyclohexylamino ordicyclopropylamino.

Alkoxyalkyl groups preferably have from 1 to 6 carbon atoms. Alkoxyalkylis, for example, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl,n-propoxymethyl, n-propoxyethyl, isopropoxymethyl or isopropoxyethyl.

Alkylthioalkyl groups preferably have from 1 to 6 carbon atoms.Alkylthioalkyl is, for example, methylthiomethyl, methylthioethyl,ethylthiomethyl, ethylthioethyl, n-propylthiomethyl, n-propylthioethyl,isopropylthiomethyl, isopropylthioethyl, butylthio-methyl,butylthioethyl or butylthiobutyl.

Cycloalkyl groups preferably have from 3 to 6 ring carbon atoms and maybe substituted by one or more methyl groups; they are preferablyunsubstituted, for example cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

Phenyl, including phenyl as part of a substituent such as phenoxy,benzyl, benzyloxy, benzoyl, phenylthio, phenylalkyl, phenoxyalkyl ortosyl, may be in mono- or poly-substituted form, in which case thesubstituents may, as desired, be in the ortho-, meta- and/orpara-position(s).

Heterocyclyl, for example, includes morpholinyl, tetrahydrofuryl.

Heteroaryl, including heteroaryl as part of a substituent such asheteroaryloxy, means a five or six member heteroaryl containing one tothree heteroatoms, each independently selected from the group consistingof oxygen, nitrogen and sulphur. It should be understood that theheteroaryl component may be optionally mono or poly substituted. Theterm heteroaryl thus includes, for example, furanyl, thiopheneyl,thiazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, isothiazolyl,pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazolyl.

Compounds of Formula Ij may contain asymmetric centres and may bepresent as a single enantiomer, pairs of enantiomers in any proportionor, where more than one asymmetric centre are present, containdiastereoisomers in all possible ratios. Typically one of theenantiomers has enhanced biological activity compared to the otherpossibilities.

Similarly, where there are disubstituted alkenes, these may be presentin E or Z form or as mixtures of both in any proportion.

Furthermore, compounds of Formula Ij comprising Q1, Q5, Q6 or Q7 or whenR¹ is hydrogen may be in equilibrium with alternative hydroxyltautomeric forms. It should be appreciated that all tautomeric forms(single tautomer or mixtures thereof), racemic mixtures and singleisomers are included within the scope of the present invention.

The skilled person will also appreciate that if n is 1 with regard toFormula Ij to form the N-oxide then the nitrogen and oxygen will becharged accordingly (N⁺O^(−).)

In a preferred embodiment of the present invention X² is oxygen.

In another preferred embodiment R¹ is selected from the group consistingof hydrogen, C₁-C₆alkyl, C₁-C₃alkoxyC₁-C₃alkyl, C₁-C₃alkoxyC₂-C₃alkoxyC₁-C₃alkyl, C₁-C₆haloalkyl, C₁-C₃alkoxy-C₁₋C₃haloalkyl andphenyl.

In another preferred embodiment R¹ is aryl, preferably phenyl, or a 5 or6-membered heteroaryl containing one to three heteroatoms eachindependently selected from the group consisting of oxygen, nitrogen andsulphur, and wherein the aryl or heteroaryl may be optionallysubstituted by one or more substituents selected from the groupconsisting of halo, C₁-C₃alkyl, C₁-C₃haloalkyl, C₁-C₃ alkoxy, C₁-C₃haloalkoxy, C₁-C₆alkyl-S(O)p-, C₁-C₆haloalkyl-S(O)p-, cyano and nitro.

In another preferred embodiment R⁵ is hydrogen.

In another preferred embodiment R⁶ is hydrogen or fluorine.

In another preferred embodiment R^(j) is selected from the groupconsisting of hydrogen, methyl and cyclopropyl.

In another preferred embodiment the herbicidal compound is of Formula(Ik):

In a more preferred embodiment of the present invention the herbicidalcompound is of Formula (Ik) wherein Q is Q1, in particular wherein A¹ isCR^(e)R^(f) and wherein R^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) arehydrogen, and wherein q=1. In another preferred embodiment of thepresent invention Q is Q1, wherein A¹ is CR^(e)R^(f) and wherein, R^(b),R^(d), R^(e) and R^(f) are hydrogen, R^(a) and R^(c) together form anethylene chain and wherein q=1

In another preferred embodiment, when the herbicidal compound is ofFormula (Ik) and wherein R⁷ is selected from the group consisting ofhydrogen, hydroxyl, halogen, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆haloalkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxy-C₂-C₆-alkoxy, C₁-C₆-alkoxy-C₁-C₆alkyl, C₁-C₆-alkoxy-C₂-C₆-alkoxy-C₁-C₆ alkyl, C₁-C₆alkylamino,C₁-C₆dialkylamino, C₂-C₆alkenylamino, C₁-C₆alkoxy-C₂-C₃-alkylamino,(C₁-C₆alkoxy-C₂-C₄-alkyl)-C₁-C₆-alkylamino, C₃-C₆ cycloalkylamino, C₃-C₆cyclohaloalkylamino, C₁-C₃alkoxy-C₃-C₆ cycloalkylamino, C₃-C₆alkynylamino and dialkylamino group in which the substituents join toform a 4-6 membered ring, optionally containing oxygen, and/oroptionally substituted by C₁-C₃-alkoxy and/or halogen, especiallyfluorine. In an even more preferred embodiment R⁷ is selected from thegroup consisting of hydrogen, chloro, methyl, ethyl, 1-methylethyl,cyclopropyl, fluoromethyl, 1-fluoro ethyl, 1,1-difluoroethyl,2,2-difluoroethyl, 1-fluoro-1-methylethyl, 2,2,2-trifluoroethyl,difluorochloromethyl, methoxy, ethoxy, methoxymethyl, 1-methoxyethyl,2-methoxyethoxy, 2-methoxyethoxymethyl, (2-methoxyethyl)amino and(2-methoxyethyl)methylamino.

In another preferred embodiment the herbicidal compound is of Formula(Im):

In another preferred embodiment of the present invention the herbicidalcompound is of Formula (Im), wherein Q is Q1, in particular wherein A¹is CR^(e)R^(f) and wherein R^(a), R^(b), R^(c), R^(d), R^(e) and R^(f)are hydrogen, and wherein q=1. In another preferred embodiment of thepresent invention Q is Q1, wherein A¹ is CR^(e)R^(f) and wherein, R^(b),R^(d), R^(e) and R^(f) are hydrogen, R^(a) and R^(e) together form anethylene chain and wherein q=1.

In another preferred embodiment wherein the herbicidal compound is ofFormula (Im) and wherein R⁷ is selected from the group consisting ofhydrogen, cyano, halogen, nitro, C₁-C₆haloalkyl, C₁-C₃alkoxyC₁-C₃haloalkyl, C₁-C₃ alkoxyC₂-C₆-alkoxyC₁-C₃ haloalkyl,C₁-C₆haloalkoxy, C₁-C₆alkylS(O)_(p),C₃₋₆cycloalkylS(O)p,C₁-C₆haloalkyl-S(O)p, C₃-C₆halocycloalkyl-S(O)p, aryl-S(O)p andheteroaryl-S(O)p. In an even more preferred embodiment R⁷ is selectedfrom the group consisting of chloro, fluoro, cyano, nitro, fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1-fluoro-1-methylethyl, difluorochloromethyl, difluoromethoxy,trifluoromethoxy, 1,1-difluoroethoxy, methylsulfinyl, methylsulfonyl,ethylsulfinyl, ethylsulfonyl, phenyl sulfinyl and phenyl sulfonyl.

In further preferred embodiments HPPD herbicidal compounds are bicycliccompounds as described in WO2009/016841.

In a particular embodiment the HPPD inhibitor is selected from the groupconsisting of benzobicyclon, mesotrione, sulcotrione, tefuryltrione,tembotrione,4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]oct-3-en-2-one,ketospiradox or the free acid thereof, benzofenap, pyrasulfotole,pyrazolynate, pyrazoxyfen, topramezone,[2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone,(2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone,isoxachlortole, isoxaflutole,α-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-chloro-benzenepropanenitrile,andα-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropanenitrile.

Other HPPD inhibitors are well known in the art and may be used withinthe methods of the present invention, including HPPD inhibitors thathave the following Chemical Abstracts registration numbers:benzobicyclon (CAS RN 156963-66-5), mesotrione (CAS RN 104206-82-8),sulcotrione (CAS RN 99105-77-8), tefuryltrione (CAS RN 473278-76-1),tembotrione (CAS RN 335104-84-2),4-hydroxy-3-[[2-(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]-bicyclo[3.2.1]oct-3-en-2-one(CAS RN 352010-68-5), ketospiradox (CAS RN 192708-91-1) or its free acid(CAS RN 187270-87-7), benzofenap (CAS RN 82692-44-2), pyrasulfotole (CASRN 365400-11-9), pyrazolynate (CAS RN 58011-68-0), pyrazoxyfen (CAS RN71561-11-0), topramezone (CAS RN 210631-68-8),[2-chloro-3-(2-methoxyethoxy)-4-(methylsulfonyl)phenyl](1-ethyl-5-hydroxy-1H-pyrazol-4-yl)-methanone(CAS RN 128133-27-7),(2,3-dihydro-3,3,4-trimethyl-1,1-dioxidobenzo[b]thien-5-yl)(5-hydroxy-1-methyl-1H-pyrazol-4-yl)-methanone(CAS RN 345363-97-5), isoxachlortole (CAS RN 141112-06-3), isoxaflutole(CAS RN 141112-29-0),α-(cyclopropylcarbonyl)-2-(methyl-sulfonyl)-β-oxo-4-chloro-benzenepropanenitrile(CAS RN 143701-66-0), andα-(cyclopropylcarbonyl)-2-(methylsulfonyl)-β-oxo-4-(trifluoromethyl)-benzenepropane-nitrile(CAS RN 143701-75-1).

The level of expression of the mutant HPPD should be sufficient toreduce substantially (relative to likewise treated plants but lackingthe mutant HPPD transgenes) the residue level of parent herbicidethroughout the plant tissue. One of ordinary skill in the art will ofcourse understand that certain mutant HPPD enzymes may confer resistanceto certain subgroups of HPPD chemistry, and one enzyme may not provideresistance to all HPPDs.

Methods of Use

The present invention further provides a method of selectivelycontrolling weeds at a locus comprising crop plants and weeds, whereinthe plants are obtained by any of the methods of the current inventiondescribed above, wherein the method comprises application to the locusof a weed controlling amount of one or more herbicides. Any of thetransgenic plants described herein may be used within these methods ofthe invention. The term “locus” may include soil, seeds, and seedlings,as well as established vegetation. Herbicides can suitably be appliedpre-emergence or post-emergence of the crop or weeds.

The term “weed controlling amount” is meant to include functionally, anamount of herbicide which is capable of affecting the growth ordevelopment of a given weed. Thus, the amount may be small enough tosimply retard or suppress the growth or development of a given weed, orthe amount may be large enough to irreversibly destroy a given weed.

Thus, the present invention provides a method of controlling weeds at alocus comprising applying to the locus a weed-controlling amount of oneor more herbicides, where the locus comprises a transgenic plant thathas been transformed with a nucleic acid molecule encoding a mutant HPPDpolypeptide or variant thereof that confers resistance or tolerance toHPPD herbicides, alone or in combination with one or more additionalnucleic acid molecules encoding polypeptides that confer desirabletraits. In one embodiment, the desirable trait is resistance ortolerance to an herbicide, including, for example, herbicides selectedfrom the group consisting of an HPPD inhibitor, glyphosate, andglufosinate. In another embodiment, the locus comprises a transgenicplant that has been transformed with any combination of nucleic acidmolecules described above, including one or more nucleic acid moleculesencoding a mutant HPPD polypeptide or variant thereof that confersresistance or tolerance to an herbicide in combination with at leastone, at least two, at least three, or at least four additional nucleicacid molecules encoding polypeptides that confer desirable traits.

In one embodiment, the present invention provides transgenic plants andmethods useful for the control of unwanted plant species in crop fields,wherein the crop plants are made resistant to HPPD chemistry bytransformation to express genes encoding mutant HPPD polypeptides, andwhere an HPPD herbicide is applied as an over-the-top application inamounts capable of killing or impairing the growth of unwanted plantspecies (weed species, or, for example, carry-over or “rogue” or“volunteer” crop plants in a field of desirable crop plants). Theapplication may be pre- or post emergence of the crop plants or of theunwanted species, and may be combined with the application of otherherbicides to which the crop is naturally tolerant, or to which it isresistant via expression of one or more other herbicide resistancetransgenes. See, e.g., U.S. App. Pub. No. 2004/0058427 and PCT App. Pub.No. WO 98/20144.

In another embodiment, the invention also relates to a method ofprotecting crop plants from herbicidal injury. In the cultivation ofcrop plants, especially on a commercial scale, correct crop rotation iscrucially important for yield stability (the achievement of high yieldsof good quality over a long period) and for the economic success of anagronomic business. For example, across large areas of the mainmaize-growing regions of the USA (the “central corn belt”), soya isgrown as the subsequent crop to maize in over 75% of cases. Selectiveweed control in maize crops is increasingly being carried out using HPPDinhibitor herbicides. Although that class of herbicides has excellentsuitability for that purpose, it can result in agronomicallyunacceptable phytotoxic damage to the crop plants in subsequent crops(“carry-over” damage). For example, certain soya varieties are sensitiveto even very small residues of such HPPD inhibitor herbicides.Accordingly, the herbicide resistant or tolerant plants of the inventionare also useful for planting in a locus of any short term carry-over ofherbicide from a previous application (e.g., by planting a transgenicplant of the invention in the year following application of an herbicideto reduce the risk of damage from soil residues of the herbicide).

The following examples are provided by way of illustration, not by wayof limitation.

EXAMPLES Example 1 Cloning, Expression and Assay of Avena-Derived HPPDSEQ ID NO:14 and Determination of kcat, Km_(HPP) and Ki (kon and koff)Values Versus Various HPPD Herbicides

The DNA sequence (SEQ ID NO:1) synthesised by GeneArt (Regensburg,Germany) encoding an HPPD derived from Avena sativa (SEQ ID NO:14) wascloned into pET24a and expressed in E. coli BL21(DE3) with 50 ng/mlkanamycin selection as described in PCT App. Pub. No. WO 02/46387.Overnight cultures grown at 30° C. were used to inoculate 3×1 litre LBin shake flasks at a ratio of 1:100. Cultures were grown at 37° C., 220rpm, until an A^(1cm) 600 nm of 0.6-0.8 was reached, the temperaturedecreased to 15° C. and induced with 0.1 mM IPTG. Cultures were grownovernight, and cells harvested after 15 min centrifugation at 10,000 g.Cells were stored at −20° C. until extraction. A cell pellet from 3litres of shake flask culture (˜12 g) was thawed in extraction buffer(50 mM Tris, 10 mM sodium ascorbate, 2 mM DTT, 2 mM AEBSF, 10 μM trypsininhibitor, 1 mM EDTA, pH 7.66) at a ratio of 1 ml buffer: 1 g cellpaste. Extract was passed through the cell disrupter at 30,000 psi, andcentrifuged at 50,000 g for 25 min. at 4° C. Optionally the extract isbuffer exchanged down Sepadex G25. Supernatants were beaded in liquidnitrogen and stored at −80° C. Levels of HPPD expression were estimatedby Western blot analysis and using purified Avena (1-10 ng) as standard.Extracts were diluted 1:6000 and 1-10 ul were loaded onto 12% SDS PAGE.In addition, expression was quantified by comparing induced anduninduced SDS PAGE with COOMASSIE® (Imperial Chemicals Industries, Ltd.,London UK) staining Gels were blotted onto PVDF membrane and Westernblots carried out using rabbit anti-wheat HPPD (1:6600) serum as primaryantibody and goat anti-rabbit FITC-linked antibodies (1:600) assecondary. Detection of bands was carried out by scanning on aFluorimager™ 595 (GE Healthcare Ltd, Buckinghamshire UK) and peakquantification was carried out by using ImageQuant™ (GE Healthcare Ltd,Buckinghamshire UK). Plasmid DNA was reisolated from all transformedstrains and the DNA sequence across the coding region confirmed.

By Western, the expression level of SEQ ID NO:14 polypeptide expressedin the E. coli extract was estimated to be about 10-14 mg/ml. out of atotal soluble protein concentration of 33.5 mg/ml.

The concentration of active HPPD in the extract was also more accuratelyestimated by active site titration. For example a range of volumes ofextract (typically 0-20 ul) were added to 50 mM BisTrisPropane buffer atpH7.0 and at 25° C. containing 25 mM Na ascorbate, 4 μg/ml bovinecatalase and 3 nmoles of ¹⁴C-labelled compound of Structure A (1.81GBq/mmol), in a total final assay volume of 425 μl.

The radiolabel protein binding reaction was quenched after 3 minutes bythe addition of 100 μl of 1 mM ‘cold’ Structure A. Protein was exchangedinto 50 mM BisTrisPropane buffer at pH 7.0 containing 0.1M KCl by rapidchromatography down a NAP5 G25 Sephadex column (GE Healthcare Ltd,Buckinghamshire UK) and ¹⁴C bound to protein fractions measured inOptiphase scintillant using a Tri-Carb 2900TR scintillation counter(Perkin Elmer, Wellesley, MA). The HPPD binding site concentration inthe extract was calculated from the titration as described in PCT PatentApp. Pub. No. WO 02/46387 and was estimated as 94.9, 78.3, and 82.3(average 85.2) μM in one extract and 47.2 μM in another example.

In an alternate method, the active site titre was calculated on thebasis of an activity-based assay titration carried out by pre-incubatingvarious ratios of extract and solutions of Structure A in order toachieve accurate titration of the active site, followed by rapiddilution into assay solution containing 100-200 μM pHPP for immediateassay by HPLC/UV quantitation of homogentisate formation after 30-40s(i.e., a time sufficiently short that inhibitor dissociation andassociation does not significantly occur on the timescale of the assay)as described below.

The Km_(HPP) and kcat values of the expressed HPPD were estimated on thebasis of assays carried out at 25° C. in solutions of 50 mMBisTrisPropane buffer at pH 7.0 containing 25 mM Na ascorbate, 4 μg/mlbovine catalase (Sigma, St. Louis, Mo.), and a range of concentrations(typically 0.5-10×Km) of 4-hydroxyphenylpyruvate. Typically assays, in afinal volume of 110 μl were started with the addition of enzyme andaccurately stopped after 20 or preferably 10 seconds with whirlimixedaddition of 20 μl 25% perchloric acid. The assay solution wastransferred to Chromacol 03-CVG HPLC vials, sealed and the amount ofhomogentisate formed in a 40 μl aliquot determined by injection onto areverse phase Aqua C18 5μ 75×4.6 mm HPLC column running 5.5%acetonitrile 0.1% TFA (Buffer A) at 1.5 ml/min. The column was eluted at1.5 ml/minute using a 2 minute wash in buffer A, followed by a 2 minutewash in a 30/70 mixture of buffer A and 100% Acetonitrile, and a further3.5 minute wash in buffer A. The elution of homogentisate was monitoredby UV at 292 nm and the amount formed in each reaction quantified bycomparison with a standard calibration curve.

Km and Vmax values were determined (for example FIG. 1) using a nonlinear least squares fit using Grafit 4™ software (Erithacus Software,Middlesex, UK). Kcat values were determined by dividing the maximumrate, Vmax expressed in nmol/second by the number of nmoles of HPPDenzyme (based on the concentration determined by active-site titration).

From one set of separate experiments similar to those that produced thedata shown in FIG. 1, on one extract of HPPD SEQ ID NO:14 the Km valuewas estimated as 6.17, 4.51, 6.09, 6.13, 4.37, 4.62, 5.41, 5.13 and 6 μM(Km average=5.38 μM). The corresponding kcat values were 4.92, 6.25,7.08, 6.26, 5.5, 6.77, 6.89, 7.12 and 7.39 s⁻¹ (kcat average=6.46 s⁻¹).Note that for this calculation and, standardly herein, Mr was taken tobe ˜94 kD and one active-site per dimer was assumed (i.e., half sitesactivity as well as inhibitor binding; see Garcia et al. (2000)Biochemistry, 39:7501-7507; Hawkes “Hydroxyphenylpyruvate Dioxygenase(HPPD)—The Herbicide Target.” In Modern Crop Protection Compounds. Eds.Kramer and Schirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp.211-220). If the alternate assumption of one active site per monomer hadbeen assumed then calculated kcat values would have been correspondinglyhalved.

On rates (governed by an association rate constant, kon) for theformation of the enzyme:inhibitor complexes, EI and off rates (governedby a dissociation rate constant, koff) were determined by methods knownin the art and essentially as described in Hawkes et al. (2001) Proc.Bright. Crop. Prot. Conf. Weeds, 2:563-568 and in PCT Patent App. Pub.No. WO 02/46387).

For example, on rates were measured by, at zero time, adding ˜60 pmolesHPPD to 50 mM BisTrisPropane buffer at pH7.0 and at 25° C. containing 25mM Na ascorbate, 4 μg/ml bovine catalase (Sigma, St. Louis, Mo.) and anexcess (˜300 pmoles) of ¹⁴C inhibitor in a total assay volume of 425 μland, at various time points (0-180 s), quenching the radiolabel bindingreaction by addition and rapid mixing of 100 μl ‘cold’ 1 mM structure A.Protein samples quenched at different times were then exchanged into 50mM BisTrisPropane buffer at pH 7.0 containing 0.1M KCl by rapidchromatography down a NAP5 G25 Sephadex column (GE Healthcare Ltd,Buckinghamshire UK) and the amount of ¹⁴C bound to protein fractionsquantified in Optiphase scintillant using a Tri-Carb 2900TRscintillation counter (Perkin Elmer, Wellesley, Mass.). The data werefit according to the scheme below in order to derive the value of theapparent second order rate constant, k2, governing the association rateof enzyme and radiolabelled inhibitor. A range of enzyme and inhibitorconcentrations were used. Optionally, the rate constant may be derivedfrom similar experiments where enzyme (at ˜0.05-0.2 μM binding sites)and, in this case, unlabelled, inhibitor (at ˜0.5 to 2 μM) are reactedfor a range of short times (0-60 s) in 50 mM BisTrisPropane buffer atpH7.0 and at 25° C. containing 25 mM Na ascorbate, 4 μg/ml bovinecatalase (Sigma, St. Louis, Mo.) and then quenched by rapid dilutioninto assay solution containing 100-200 μM HPP for immediate assay byHPLC/UV quantitation of homogentisate formation after 30-40 s (i.e., atime sufficiently short that inhibitor dissociation and association doesnot significantly occur on the timescale of the assay) as describedabove. Further example methods are described in PCT Patent App. Pub. No.WO 02/46387.

Off rates (k1 in the scheme below) were derived from exchange ratestudies where either the test inhibitor, I, or its exchange partner, Jwere radiolabelled and the data fit according to the scheme below. Asnoted in Hawkes et al. (2001) Proc. Bright. Crop. Prot. Conf. Weeds,2:563-568, HPPD preparations typically appear to contain 15-30% of amore rapidly exchanging (weaker binding) fraction of inhibitor bindingsites. This may be a slightly damaged form of the enzyme (it maintainscatalytic activity and may have a higher substrate Km) and, except whereoff rates are so fast that fast and slow exchanging fractions arerendered indistinguishable, off rates always refer to the behaviour ofthe majorly slower exchanging fraction that represents 70-85% bulk ofthe HPPD inhibitor binding sites present in the extracts tested.

Off rates were determined by preincubating, for example, ˜200 pmoles ofHPPD binding sites (determined as described above by active sitetitration in a 3 min reaction with structure A) in 50 mM BisTrisPropanebuffer at pH 7.0 and at 25° C. containing 25 mM Na ascorbate, 4 μg/mlbovine catalase (Sigma, St. Louis, Mo.) containing ˜1.0 nmole ¹⁴Cinhibitor @ 25° C. in a total assay volume of 1.3 mls. After 30 minutesthe exchange reaction was initiated with addition of 100 μl 1 mM ‘cold’structure A with thorough mixing, and, immediately, 150 μl werewithdrawn and loaded onto a NAP5 column, the protein exchanged into 50mM BisTrisPropane buffer at pH 7.0 containing 0.1M KCl by rapid (<2 min)chromatography down a NAP5 G25 Sephadex column (GE Healthcare Ltd,Buckinghamshire UK) and the amount of ¹⁴C bound to protein measured byOptiphase scintillant using a Tri-Carb 2900TR scintillation counter(Perkin Elmer, Wellesley, Mass.). Further aliquots were removed andmeasured in the same way at various times over minutes or hours asrequired in order to determine the exchange kinetics.

In one variant of the method useful to better distinguish between offrates that were relatively rapid (e.g., where t ½<15 min at 25° C.) thetemperature of the experiment was reduced from 25° C. to icetemperature. In this case, off rates were determined by preincubating˜200 pmoles HPPD in reaction buffer (50 mM BTP pH7, 25 mM Na ascorbate,4 ug/ml bovine catalase, and 10% glycerol) containing ˜1.0 nmoles ¹⁴Cinhibitor at 25° C. in a total assay volume of 1.3 mls. After 30 minutesthe reaction vessel was transferred to ice. After a further 10 minutesat ice temperature the exchange reaction was initiated by addition of100 μl 1 mM Structure A, with thorough mixing, and 150 μl was withdrawnand loaded onto a NAP5 column in a cold room at ˜5-8° C. in order toquantify the amount of radiolabel remaining bound to the protein atvarious time from the start of exchange at ice temperature.

Off rates (k1) of HPPD inhibitors that are not available radiolabelledor that present other measurement problems (for example high levels ofbackground non-specific protein-binding which can be measured asradiolabel binding that persists in the presence of high concentrationsof ‘cold’ inhibitor) may be measured indirectly. In this case the enzymecomplex (˜0.1-0.2 μM) is first formed with the unlabelled inhibitor andthen the exchange kinetics derived by chasing it off with high aconcentration of ¹⁴C-labelled structure A and monitoring the rate atwhich the label becomes bound to protein. Structure A is a particularlypotent inhibitor with known kinetics and in a 20 fold or more excesswill, in equilibrium, >95% occupy the binding sites in exchangecompetition with the other inhibitors tested here and indeed most otherinhibitors (those skilled in the art will of course design theexperiment/relative concentrations and fit the data accordingly).Exemplary methods are also described in PCT Patent App. Pub. No. WO02/46387.

Exemplary on and off rate data (and derived Ki values) were obtained forthe Avena-derived HPPD SEQ ID NO:11 for the following compounds asfollows.

Off rate (k1=1.67E-05 s⁻¹). 25° C., direct, radiochemical method.

On rate (k2=8.50E+04 M⁻¹ s⁻¹). 25° C., direct radiochemical method.

Kd=1.96E-10 M.

Kd/Km ratio=0.000036

Off rate k1(av)=8.1 E-04 s⁻¹ at 25° C. (individual experiments yieldedk1=8.00E-04, 8.88E-04, 7.50E-04 and 8.00E-04 as determined by thedirect, radiochemical method). Measured at ice temperature k1=1.21E-05s⁻¹ (individual experiments yielding 1.16E-05 s⁻¹, 1.0E-05 s⁻¹, 1.2E-05s⁻¹, 1.5E-05 s⁻¹) by the direct, radiochemical method.

On rate k2(av)=6.7E+04 s⁻¹ M⁻¹ at 25° C. (individual experiments yieldedk2=6.35E+04, 7.50E+04, 6.2E+04 as determined by the direct radiochemicalmethod). For mesotrione which has a relatively fast off rate estimatesfor on rate based on the activity-based method were more variableranging from 4.2E+04 s⁻¹ M⁻¹, 4.9E+04 s⁻¹ M⁻¹ to 7.5 E+04 s⁻¹ M⁻¹ at 25°C.

Kd was thus estimated from the radiochemical data as 1.16E-08 Mcorresponding to a Kd/Km ratio of 0.00217.

Off rate k1(av)=7.04 E-05 s⁻¹ at 25° C. (individual experiments yieldedk1=7.80E-05, 9.17E-05, 4.5E-05, 6E-05, 7 E-05 and 7.80E-05 as estimatedby the indirect radiochemical method).

On rate k2=7.50E+03 s⁻¹ M⁻¹ at 25° C. as estimated by the directradiochemical method is in good agreement with estimates from the enzymeactivity-based method of 7.50E+03 s⁻¹ M⁻¹, 7.80E+03 s⁻¹ M⁻¹, 7.60E+03s⁻¹ M⁻¹, 7.20E+03 s⁻¹ M⁻¹ and 1.0E+04 s⁻¹ M⁻¹ at 25° C.

Based on the radiochemical method the estimate of Kd=9.4 E-09M.

Therefore the estimate of Kd/Km ratio is then=0.0017.

Off rate k1=3.96E-05 s⁻¹ at 25° C. as determined using the direct,radiochemical method (individual measurements of 4.17E-05 s⁻¹ and3.75E-05 s⁻¹).

On rate k2=3.20E+04 M⁻¹ s⁻¹ at 25° C. as determined by the directradiochemical method. This is in fair agreement with estimates from theactivity based method for on rate of 3.20E+04 M⁻¹ s⁻¹ and 5.7E+04 M⁻¹s⁻¹.

Based on the radiochemical methods the estimate of Kd=1.23E-9 M.

The estimate of Kd/Km ratio=0.00023.

Off rate k1=4.17E-05 s⁻¹ at 25° C. as determined by the indirect,radiochemical method. (individual measurements of 5.50E-05 s⁻¹ and2.85E-05 s⁻¹).

On rate k2=1.30E+05 M⁻¹ s⁻¹ at 25° C. as determined by the directnon-radiochemical method.

The estimate of Kd=3.21E-10M.

The estimate of Kd/Km ratio=0.000059.

Example 2 Cloning, Expression and Assay of Further Variants ofAvena-Derived HPPDs SEQ ID NOS:12-20 and Determination of kcat, Km_(HPP)and Ki (kon and koff) Values Versus Various HPPD Herbicides

DNA sequences corresponding to SEQ ID NOS:2-14, encoding HPPDpolypeptides corresponding to SEQ ID NOS:15-26 derived from Avenasativa, were synthesized by GeneArt (Regensburg, Germany), cloned intopET24a, and expressed in E. coli BL21(DE3) with 50 ng/ml kanamycinselection as described in PCT App. Pub. No. WO 02/46387. Cells weregrown, protein extracts were prepared, and HPPD active site titres andkinetic measurements (of kcat, KmHPP, k1, k2 and Ki values) were carriedout as described in Example 1.

Within the present example, the following HPPD sequences were used:

HPPD SEQ ID NO:15 was changed relative to SEQ ID NO:14 by thesubstitution of A for Q within the sequence motif GVQHIA (residues 1-6of SEQ ID NO:55).

HPPD SEQ ID NO:16 was changed relative to SEQ ID NO:14 by thesubstitution of G for Q within the sequence motif GVQHIA (residues 1-6of SEQ ID NO:55).

HPPD SEQ ID NO:17 was changed relative to SEQ ID NO:14 by thesubstitution of S for Q within the sequence motif GVQHIA (residues 1-6of SEQ ID NO:55).

HPPD SEQ ID NO:18 was changed relative to SEQ ID NO:14 by thesubstitution of T for 1 within the sequence motif SQIQTY (residues 1-6of SEQ ID NO:53).

HPPD SEQ ID NO:19 was changed relative to SEQ ID NO:14 by thesubstitution of A for 1 within the sequence motif SQIQTY (residues 1-6of SEQ ID NO:53).

HPPD SEQ ID NO:20 was changed relative to SEQ ID NO:14 by thesubstitution of S for 1 within the sequence motif SQIQTY (residues 1-6of SEQ ID NO:53).

HPPD SEQ ID NO:21 was changed relative to SEQ ID NO:14 by thesubstitution of V for 1 within the sequence motif SQIQTY (residues 1-6of SEQ ID NO:53).

HPPD SEQ ID NO:22 was changed relative to SEQ ID NO:14 by thesubstitution of M for L within the sequence motif SGLNS (residues 5-9 ofSEQ ID NO:43).

HPPD SEQ ID NO:23 was changed relative to SEQ ID NO:14 by thesubstitution of W for A within the sequence motif FAEFT (residues 5-9 ofSEQ ID NO:42).

HPPD SEQ ID NO:24 was changed relative to SEQ ID NO:14 by thesubstitution of M for L within the sequence motif G(I,V)LVDRD (SEQ IDNO:30).

HPPD SEQ ID NO:25 was changed relative to SEQ ID NO:14 by thesubstitution of A for L within the sequence motif G(I,V)LVDR (residues1-6 of SEQ ID NO:30).

HPPD SEQ ID NO:26 was changed relative to SEQ ID NO:14 by thesubstitution of M for L within the sequence motif G(I,V)LVDR (residues1-6 of SEQ ID NO:30) and by the substitution of M for L within thesequence motif SGLNS (residues 5-9 of SEQ ID NO:43).

Values (generally radiochemically determined) of kon (k2), koff (k1),and Ki (all at 25° C.) were obtained for the HPPDs in the presentexample versus the various inhibitor structures as shown in Table 3. Thevalues given for the reference SEQ ID NO:14 in Table 3 are the averagevalues from a number of experiments as described above. All of theexperiments with the other HPPDs included side by side measurements withSEQ ID NO:14 as a comparative control. Within experiments, the ratios ofon and off rates relative to this side by side control were reproducibleeven where absolute values varied somewhat. Thus the values given inTable 3 for HPPD SEQ ID NOs:15-26 are normalized versus the averagecontrol values for HPPD SEQ ID NO:14 according to these observed ratios.

TABLE 3 Summary of Values of kon, koff and Kd for HPPD Variants kon(k2)/Kd kon(k2)/ Kd HPPD variant s/M koff(k1)/s nM s/M koff(k1)/s nMStructure A Structure B SEQ ID# 14 85000 1.67E−05 0.20 67000 8.10E−0411.6 SEQ ID# 15 35000 3.33E−05 0.95 70000 8.00E−04 11.4 SEQ ID# 16 ND NDND 53000 2.00E−03 37.7 SEQ ID# 17 ND ND ND 53000 1.00E−03 18.9 SEQ ID#18 42000 1.67E−05 0.40 35000 6.00E−04 17.1 SEQ ID# 19 ND ND ND 380007.50E−04 19.7 SEQ ID# 20 ND ND ND 31500 9.00E−04 28.6 SEQ ID# 21 850001.67E−05 0.20 70000 6.00E−04 8.6 SEQ ID# 22 85000 1.08E−05 0.13 700001.20E−03 17.1 SEQ ID# 23 85000 2.83E−05 0.33 70000 7.00E−04 10.0 SEQ ID#24 85000 2.30E−05 0.27 70000 1.57E−03 22.4 SEQ ID# 25 ND ND ND 200008.00E−04 40.0 SEQ ID# 26 ND ND ND 70000 3.00E−03 42.9 Structure CStructure D SEQ ID# 14 7500 7.04E−05 9.4 32000 3.96E−05 1.2 SEQ ID# 157500 1.13E−04 17.7 ND 2.37E−05 ND SEQ ID# 16 4500 1.20E−04 26.7 ND ND NDSEQ ID# 17 9400 6.65E−05 7.1 ND ND ND SEQ ID# 18 7500 9.00E−05 11.9 ND3.96E−05 ND SEQ ID# 19 7500 6.60E−05 8.9 ND ND ND SEQ ID# 20 101006.60E−05 6.6 ND ND ND SEQ ID# 21 7500 9.00E−05 11.9 ND 3.96E−05 ND SEQID# 22 4400 9.00E−05 23.9 ND 2.37E−05 ND SEQ ID# 23 7500 ND ND ND2.37E−05 ND SEQ ID# 24 4900 7.82E−05 16.0 32000 9.18E−05 2.9 SEQ ID# 254800 1.13E−04 23.0 ND ND ND SEQ ID# 26 ND 9.00E−05 ND ND ND ND

For example, the off rate of mesotrione (structure B) from HPPD SEQ IDNO:14 was clearly differentiated from that of SEQ ID NO:24 (see FIGS.4A-4C) with the goodness of fits being sensitive to small changes inkoff. From these data it can be seen that mesotrione dissociated abouttwice as fast from HPPD SEQ ID NO:26 as from HPPD SEQ ID NO:24, and fromHPPD SEQ ID NO:24 about twice as fast as from HPPD SEQ ID NO:14.Generally the absolute estimates of koff obtained from the fits to thedata were reproducible to within +/−10% and usually better.

When off rates became relatively fast (t½<10 minutes) it was also usefulto make comparative measurements at ice temperature in order to moreaccurately confirm the differential between one HPPD and another. Thus,for example, at ice temperature, mesotrione dissociation from HPPD SEQNO:14 was governed by a rate constant, koff, of 1.16E-05 s⁻¹ (muchslower than the value of 8.1 E-04 s⁻¹ estimated at 25° C.) whereas forSEQ ID NOS:22, 24 and 26, the corresponding mesotrione off rates at icetemperature were 2.17E-05 s⁻¹, 2.25E-05 s⁻¹ and 4.17E-05 s⁻¹; thesevalues being in good proportionate agreement with those at 25° C. (SeeTable 3).

A number of conclusions were derived from the data in Table 3. Theproperties of HPPDs SEQ ID NOS:15-17 indicated that certainsubstitutions for asparagine(Q) within the amino acid sequence GVQHIprovided significant improvements relative to HPPD SEQ ID NO:14 intolerance (slower values of kon and/or faster values of koff), withrespect to, for example, Structures A, B and C.

Data from HPPDs SEQ ID NOS:18-21 indicated that certain substitutionsfor isoleucine(I) within the amino acid sequence SQIQTY providedsignificant improvements relative to HPPD SEQ ID NO:14 in tolerance(mainly via slower values of kon), with respect to, for example,Structures A and B.

Data from HPPD SEQ ID NO:22 indicated that certain substitutions forleucine(L) within the amino acid sequence ESGLN provided significantimprovements relative to HPPD SEQ ID NO:14 in tolerance (mainly viafaster values of koff) with respect to, for example, Structures B and C.

Data from HPPD SEQ ID NO:23 indicated that certain substitutions foralanine (A) within the amino acid sequence EFAEF provided significantimprovements relative to HPPD SEQ ID NO:14 in tolerance (mainly viafaster values of koff) with respect to, for example, Structure A.

Data from HPPDs SEQ ID NOS:24 and 25 indicated that certainsubstitutions for leucine (L) within the amino acid sequence G(I,V)LVDRDprovided significant improvements relative to HPPD SEQ ID NO:14 intolerance (via faster values of koff and/or slower values of kon) withrespect to, for example, Structure A, Structure B, Structure C andStructure D.

Data from HPPD SEQ ID NO:26 indicated that the combination of certainsubstitutions for leucine(L) within the amino acid sequence ESGLN withcertain substitutions for leucine (L) within the amino acid sequenceG(I,V)LVDRD provided yet further significant improvements relative toHPPD SEQ ID NO:14 (and over and above the effect of either singlechange) in tolerance (mainly via faster values of koff) with respect to,for example, Structures B.

Again, as described for Example 1, kcat and Km values were determinedfor a number of the HPPDs of the invention expressed in extracts and thevalues are depicted in Table 4.

TABLE 4 Km and kcat Values of Various HPPDs Km kcat kcat/Km HPPD variantuM s−1 uM−1s−1 SEQ ID #14 5.38 6.46 1.2 SEQ ID #18 35.98 17.94 0.50 SEQID #21 5.98 5.47 0.91 SEQ ID #22 12.43 5.79 0.46 SEQ ID #24 4.74 4.350.92 SEQ ID #26 10.58 4.05 0.38

A number of the HPPD variants had low Km values similar to HPPD SEQ IDNO:14 and higher values of Ki/Km with respect to the various HPPDherbicides and, thus, overexpression in plants expected to provideenhanced herbicide tolerance to these herbicides. For example, HPPD SEQID NO:24 was twice as resistant to mesotrione as was HPPD SEQ ID NO:14since it exhibited a Ki/Km ratio of 0.0047 as compared with 0.0021.

In addition, all of the above sequences as well as libraries of variantsmutated at the same amino positions that showed altered and enhancedlevels of herbicide tolerance are useful to be included in mutagenesisand shuffling processes in order to generate yet further shuffled andmutated HPPDs useful as transgenes for conferring herbicide tolerance.For example, the mutants disclosed in Table 5 are useful for generatinga herbicide tolerant HPPD mutant polypeptide and for inclusion inrecombination reactions to generate further HPPDs.

TABLE 5 Examples of Mutations Useful in HerbicideTolerant HPPD Polypeptides Amino acid region of Mutation SEQ ID NO: 14K411L GGFG

GNFS K411T GGFG

GNFS K411S GGFG

GNFS K411M GGFG

GNFS K411A GGFG

GNFS K411E GGFG

GNFS K411V GGFG

GNFS M325L GFEF

APPQ L271I VLLP

NEPV L271M VLLP

NEPV L271V VLLP

NEPV G408A GGCG

FGKG G408S GGCG

FGKG G408T GGCG

FGKG V258M GLNS

VLAN V258I GLNS

VLAN V258A GLNS

VLAN V258K GLNS

VLAN V217I RFDH

VGNV V217A RFDH

VGNV V217M RFDH

VGNV V217C RFDH

VGNV L271I VLLP

NEPV L271M VLLP

NEPV L271V VLLP

NEPV A326S FEFM

PPQA A326K FEFM

PPQA A326P FEFM

PPQA A326D FEFM

PPQA A326R FEFM

PPQA A326N FEFM

PPQA A326Y FEFM

PPQA A326H FEFM

PPQA I370V VLLQ

FTKP Y287F QIQT

LEYH G254S TTES

LNSV G254A TTES

LNSV E416Q GNFS

LFKS I339L GVRR

AGDV L269M EAVL

PLNE L269F EAVL

PLNE S420A ELFK

IEDY I372S LQIF

KPVG Y172V EVEL

GDVV I299M GVQH

ALAS

As another example, the mutants disclosed in Table 6 are also useful forgenerating a herbicide tolerant HPPD mutant polypeptide and forinclusion in recombination reactions to generate further HPPDs.

TABLE 6 Examples of Mutations Useful in HerbicideTolerant HPPD Polypeptides Amino acid region of Mutation SEQ ID NO: 14K411L GGFG

GNFS K411T GGFG

GNFS K411S GGFG

GNFS K411M GGFG

GNFS K411A GGFG

GNFS K411E GGFG

GNFS K411V GGFG

GNFS M325L GFEF

APPQ L271I VLLP

NEPV L271M VLLP

NEPV L271V VLLP

NEPV G408A GGCG

FGKG G408S GGCG

FGKG G408T GGCG

FGKG V258M GLNS

VLAN V258I GLNS

VLAN V258A GLNS

VLAN V258K GLNS

VLAN V217I RFDH

VGNV V217A RFDH

VGNV V217M RFDH

VGNV V217C RFDH

VGNV L271I VLLP

NEPV L271M VLLP

NEPV L271V VLLP

NEPV A326S FEFM

PPQA A326K FEFM

PPQA A326P FEFM

PPQA A326D FEFM

PPQA A326R FEFM

PPQA A326N FEFM

PPQA A326Y FEFM

PPQA A326H FEFM

PPQA I370V VLLQ

FTKP Y287F QIQT

LEYH G254S TTES

LNSV G254A TTES

LNSV E416Q GNFS

LFKS I339L GVRR

AGDV L269M EAVL

PLNE L269F EAVL

PLNE S420A ELFK

IEDY I372S LQIF

KPVG Y172V EVEL

GDVV I299M GVQH

ALAS

Table 7 summarises data from kinetic studies of a range of mutants ofHPPD SEQ ID NO:14 expressed relative to the control, ‘none’, meaningnon-mutated HPPD SEQ ID NO:14. Experiments were carried out as describedfor Table 4. ‘Sulc’ denotes sulcotrione and ‘nd’ means ‘no data’. ForV217I, L271I, L271V, V258M and A326R, the relative values of kcat wereestimated from comparisons of the initial rates in cell extracts ofsimilarly prepared and expressed HPPDs in conventional enzyme activityassays at pH 7.0, 25° C. and at a substrate concentration of 120 μM HPP.V217I, V258M and A326R, M325L and L358M mutants of SEQ ID NO:14 areactive HPPD enzymes that offer some resistance to sulcotrione, and mayalso offer resistance to B. K411T offers significant resistance to E andespecially since the greater than 5× increase in Kd to this herbicidecomprises mainly an improvement in off rate (3.5×) rather than in onrate. L358M, M325L and K411T all offer improvements with respect to D.For herbicide tolerance L271I and L271V appear to offer significantadvantages in kcat over unmutated enzyme.

TABLE 7 Relative Kinetics of Various Mutants of SEQ ID NO: 14 chemical Bsulc C D E Rate mutation koff Kd Kd koff Kd koff Kd koff Kd kcat kcat/Kmnone 1 1 1 1 1 1 1 1 1 1 1 L358M 2 2 2 1.1 1.7 2.3 2.3 1.2 1.5 0.7 0.8M325L 1.1 1.1 1.3 1 1 1.2 1.4 1 1 1.2 1.3 V217I 1.5 1.5 1.3 1.1 1.1 1 11 1 1.1 1.1 V258M nd Nd 1.2 nd nd nd nd nd nd 1 nd L281I nd Nd 0.8 nd ndnd nd nd nd 1.7 nd L281V nd Nd 0.6 nd nd nd nd nd nd 2 nd A326R 1.7 1.71.6 0.9 0.9 1.4 1.4 1.3 1.6 1.2 1.4 K411T 0.3 0.5 0.9 1 1.1 1.2 3.6 3.55.4 1 0.4

It will be appreciated that the majority of substitutions to amino acidswithin the highly conserved active-site region of HPPD and that liewithin 8° A of the atoms of bound mesotrione (according tointerpretation of published X Ray crystallographic data of the maize andarabidopsis HPPDs and homology model building to oat HPPD) result indisabled or only partially functional enzymes. From sequence alignmentsof (active) HPPD sequences in the database, about 60 single or doublemutants of SEQ ID NO:14 were selected as amenable to changes in someresidues without loss of enzyme activity (on the basis that they werechanges that represented some of the spread of sequence variation foundamongst natural HPPDs at these positions). These mutants were made,grown, the HPPDs expressed and extracts prepared and tested for theircatalytic activity and resistance to mesotrione (relative to thecontrol, unmutated SEQ ID NO:14). Even amongst this privileged set themajority exhibited significantly impaired catalytic activity and/or weresignificantly more sensitive to sulcotrione than the control. Y287F andI370V were neutral mutations with similar (within 20%) values of kcatand resistance to sulcotrione as the unmutated enzyme. Amongst a furtherset of about 70 mutants encompassing residues as far as 10° A from theatoms of the bound inhibitors further such neutral mutations (withrespect to SEQ ID NO:14) were G254S, G254A, E416Q, V258M, V258I, V258A,V258K, S415K, 5415Q, I421L, A326S, L269M, L269F, S420A, T372S, Y172V and1299M. These further mutations can all be optionally combined with theresistance conferring mutations to produce catalytically active variantsof HPPD herbicide resistant enzymes of the current invention.

A further mutant of HPPD SEQ ID NO:14, G408A, exhibited inhibitionkinetics in respect of B and C showing that this mutant was relativelyresistant to inhibition by these compounds. The timecourses ofinhibition were not straightforward and could not be fit to the kineticmodel described above. The experimental method used was similar to thatdescribed above for measuring inhibitor-binding on rates by monitoringenzyme activity. The time courses of inhibition are depicted in FIGS.10A-10D. Enzyme at about 75 nM was incubated with inhibitor at 0.15 or0.6 μM for various times up to 260s and then immediately assayed over a150s period following addition of 115 μM HPP (and thus with [S] at˜30×Km also dramatically slowing any further inhibitor binding). In thecase of the mutant there appeared to be an initial rapid phase ofinhibition which then slowed to leave the enzyme only partly inhibited.In the case of control enzyme inhibition proceeded to (or was on coursetowards) full inhibition. Although note that in the case of inhibitionof the control enzyme by compound B did not quite reach 100%. The ˜8%residual activity in this case was an artifact of the method due to therelatively fast off rate of compound B which allowed some activity torecover during the 150s assay used to monitor the progress of thereaction between enzyme and inhibitor. This artifact is negligible withslower dissociating inhibitors such as C. Over the time of theexperiment and at 0.6 μM B, inhibition of mutant G408A appeared to leveloff to a residual activity of about 35%. It appeared that this was notdue to an even faster off rate for B from G208A than from the controlenzyme since, at ice temperature, the radiochemically determined offrate of B from G408A appeared indistinguishable from the rate observedwith the control SEQ ID NO:14 HPPD. Mutant G408 also exhibited a similarkcat and kcat/Km to SEQ ID NO: 14 HPPD. Whatever the explanation both Band C appeared to inhibit the G408A mutant HPPD to a lesser extent thanthe control enzyme. It is also notable the G408A activity appearedunstable since the control activity in the absence of inhibitor declinedover the course of the experiment. The addition of inhibitor appeared toarrest this decline in activity and in further experiments it wasconfirmed that mutant G408A activity was unstable in the absence ofinhibitor or substrate but was stabilized by inhibitor and appeared noless stable than wild type enzyme over extended assay time courses inthe presence of substrate or when partially inhibited by HPPD herbicide.Thus, despite some instability, mutant G408A is useful alone or incombination with other mutations to provide useful herbicide tolerancewhile herbicide is present in the plant tissues where it is expressed.

Aside from enzyme kinetic experiments, enhanced resistance to HPPDherbicides was further demonstrated when the HPPDs of the currentinvention were expressed in E. coli and the comparative herbicideresistances of the various HPPDs assessed visually via the production ofpyomelanin. For example HPPD SEQ ID NO:14 and HPPD SEQ ID NO:24 wereexpressed from a pET24 vector in E. coli BL21 cells. Grown withoutaddition of IPTG there was sufficient expression of HPPD for cultures toslowly turn brown due to the production of pyomelanin pigment (whichresults from auto-oxidation of HPPD-derived homogentisate). Cells weregrown from a 10% starting inoculum into 0.5 ml of L-broth containing 50μg of kanamycin ml⁻¹ in 45 well plates for 48-96 h at 15° C. Typicallypyomelanin colour in the medium was read (at 430 nm) after ˜72 h. It wasnoted that addition of 12.5 ppm mesotrione caused significantlyproportionately less inhibition of pyomelanin colour development in thecells expressing HPPD SEQ ID NO:24 than expressing HPPD SEQ ID NO:14.FIG. 5 compares the absorbance of the medium obtained after 72 h in sideby side triplicate grows of E. coli expressing HPPD SEQ ID NOS:14, 18,and 24 all grown in the same plate.

Cells expressing HPPD SEQ ID NO:24, which exhibited the highest ratio ofKi/Km, consistently exhibited the least difference in colour betweencells grown with and without 12.5 ppm mesotrione present in the medium.The same was seen when the mesotrione was replaced with 20 ppmsulcotrione (data not shown) indicating that SEQ ID NO 24 offersenhanced tolerance to sulcotrione as well as to mesotrione. Similarly,cells expressing mutant G408A also exhibited resistance relative to HPPDSEQ ID NO:14 to sulcotrion according to the pyomelanin assay with 25 ppmsulcotrione.

Example 3 Preparation and Testing of Stable Transgenic Plant LinesExpressing a Heterologous HPPD Enzyme

In the present example, mutant HPPD genes derived from Avena HPPD werethe sequences set forth in SEQ ID NOS:1-13 (optimized for tobacco) or,optionally, are codon optimized according to target crop (e.g., soybean)and prepared synthetically and obtained commercially from GeneArt(Regensburg, Germany). Each sequence is designed to have 5′ NdeI and3′BamHI sites to facilitate direct cloning. For example, the sequencesset forth in SEQ ID NOS:11, 12, or 13 are cloned into a suitable binaryvector for Agrobacterium-based plant transformation.

In a particular example genes encoding HPPD SEQ ID NO:14 and HPPD SEQ IDNO: 24 were cloned into identical expression constructs as describedbelow and transformed into tobacco.

As described in PCT Patent App. Pub. No. WO 02/46387, the HPPD encodingnucleotide sequence is edited by PCR (or initially synthesized) toinclude 5′ Nco 1 and 3′ Kpn 1 sites (and to remove any such internalsites). This product is then ligated into pMJB1. pMJB1 was a pUC19derived plasmid which contains the plant operable double enhancedCaMV35S promoter; a TMV omega enhancer, and the NOS transcriptionterminator. A schematic representation of the resulting plasmid is shownin FIG. 2 of PCT Patent App. Pub. No. WO 98/20144. The expressioncassette, comprising the double enhanced 35S promoter, TMV omega leader,4-HPPD gene and nos terminator, is excised using Hind III/Eco R1(partial Eco R1 digest) and cloned into similarly digested pBIN 19 andtransformed into E. coli TOP 10 competent cells. DNA recovered from theE. coli is used to transform Agrobacterium tumefaciens LBA4404, andtransformed bacteria are selected on media contain rifampicin andkanamycin. Tobacco tissue is subjected to Agrobacterium-mediatedtransformation using methods well described in the art or as describedherein. For example, a master plate of Agrobacterium tumefacienscontaining the HPPD expressing binary vector is used to inoculate 10 mlLB (L broth) containing 100 mg/1 Rifampicin plus 50 mg/1 Kanamycin usinga single bacterial colony. This is incubated overnight at 28° C. shakingat 200 rpm. This entire overnight culture is used to inoculate a 50 mlvolume of LB containing the same antibiotics. Again this is culturedovernight at 28° C. shaking at 200 rpm. The Agrobacterium cells arepelleted by centrifuging at 3000 rpm for 15 minutes and then resuspendedin MS (Murashige and Skoog) medium containing 30 g/1 sucrose, pH 5.9 toan OD (600 nM)=0.6. This suspension is dispensed in 25 ml aliquots intopetri dishes.

Clonally micro-propagated tobacco shoot cultures are used to exciseyoung (not yet fully expanded) leaves. The mid rib and outer leafmargins are removed and discarded, and the remaining lamina cut into 1cm squares. These are transferred to the Agrobacterium suspension for 20minutes. Explants are then removed, dabbed on sterile filter paper toremove excess suspension, then transferred onto solid NBM medium (MSmedium containing 30 g/1 sucrose, 1 mg/1 BAP (benzylaminopurine) and 0.1mg/1 NAA (napthalene acetic acid) at pH 5.9 and solidified with 8 g/1Plantagar), with the abaxial surface of each explant in contact with themedium. Approximately 7 explants are transferred per plate, which arethen sealed and maintained in a lit incubator at 25° C. for a 16 hourphotoperiod for 3 days.

Explants are then transferred onto NBM medium containing 100 mg/1Kanamycin plus antibiotics to prevent further growth of Agrobacterium(200 mg/1 timentin with 250 mg/1 carbenicillin). Further subculture ontothis same medium was then performed every 2 weeks.

As shoots start to regenerate from the callusing leaf explants, theseare removed to Shoot elongation medium (MS medium, 30 g/1 sucrose, 8 g/1Plantagar, 100 mg/1 Kanamycin, 200 mg/1 timentin, 250 mg/1carbenicillin, pH 5.9). Stable transgenic plants readily root within 2weeks. To provide multiple plants per event to ultimately allow morethan one herbicide test per transgenic plant, all rooting shoots aremicropropagated to generate 3 or more rooted clones.

Putative transgenic plants that are rooting and showing vigorous shootgrowth on the medium incorporating Kanamycin are analysed by PCR usingprimers that amplified a 500 bp fragment within the HPPD transgene.Evaluation of this same primer set on untransformed tobacco showedconclusively that these primers would not amplify sequences from thenative tobacco HPPD gene.

Transformed shoots are divided into 2 or 3 clones and regenerated fromkanamycin resistant callus. Shoots are rooted on MS agar containingkanamycin. Surviving rooted explants are re-rooted to provideapproximately 70-80 kanamycin resistant and PCR-positive events fromeach event.

Once rooted, plantlets are transferred from agar and potted into 50%peat, 50% John Innes Soil No. 3 with slow-release fertilizer in 3 inchround pots and left regularly watered to establish for 8-12d in theglass house. Glass house conditions are about 24-27° C. day; 18-21° C.night and approximately a 14 h photoperiod. Humidity is adjusted to ˜65%and light levels used are up to 2000 μmol/m² at bench level.

Two transgenic populations each of about 80 tobacco plants andcomprising, alternatively, an HPPD gene encoding HPPD SEQ ID NO:14 orHPPD SEQ ID NO:24 within otherwise identical expression cassettes werethus produced. These two populations were grown on until about the 2-4leaf stage and then each divided into two subpopulations, one comprisingthose plantlets that had emerged rather larger and more advanced fromtissue culture and the other population comprising the smaller plants.Thus the small sized populations of SEQ ID NO:14 and SEQ ID NO:24appeared visually similar to comparable each other as did the twopopulations of larger sized plants.

The two smaller populations were each then sprayed with 300 g/ha ofmesotrione and the two larger populations with 500 g/ha. Callisto® wasmixed in water with 0.2-0.25% X-77 surfactant and sprayed from a boom ona suitable track sprayer moving at 2 mph with the nozzle about 2 inchesfrom the plant tops. Spray volume was 2001/ha.

Plants were assessed for damage and scored at 13 days after treatment(DAT). All four populations appeared highly resistant to the herbicidetreatments but the SEQ ID NO:24 HPPD expressing populations more so thanthe control SEQ ID NO:14 populations. From the two larger-sizedpopulations sprayed with 500 g/ha only 4 of 38 (10%) morphologicallynormal PCR positive plants (one emerged chimeric) expressing SEQ IDNO:24 exhibited symptoms of herbicide damage whereas 9 out of a total of33 (27%) of SEQ ID NO:14 expressing plants exhibited damage. There waslittle damage to see on the two smaller-sized populations sprayed with300 g/ha mesotrione; here 2 of 28 SEQ ID NO:24 expressing plantsexhibited visible herbicide damage as compared with 4 of 26 SEQ ID NO:14expressing plants.

Plants of events showing the least damage are grown to flowering, thenbagged and allowed to self. The seed from selected events are collectedand sown again in pots, and tested again for herbicide resistance in aspray test for resistance to HPPD herbicide (for example mesotrione).Single copy events amongst the T1 plant lines are identified by their3:1 segregation ratio (with respect to kanamycin and/or herbicide) andby quantitative RT-PCR. Seed from the thus selected T1 tobacco (var.Samsun) lines are sown in 3 inch diameter pots containing 50% peat and50% John Innes Soil No. 3.

Example 4 Construction of Soybean Transformation Vectors

Binary vectors for dicot (soybean) transformation were constructed witha promoter, such as a synthetic promoter containing a CaMV 35S and anFMV transcriptional enhancer and a synthetic TATA box driving theexpression of an HPPD coding sequence, such as SEQ ID NO:24, followed byNos gene 3′ terminator. The HPPD gene was codon-optimized for soybeanexpression based upon the predicted amino acid sequence of the HPPD genecoding region. In the case that HPPD itself was not used as theselectable marker, Agrobacterium binary transformation vectorscontaining an HPPD expression cassette were constructed by adding atransformation selectable marker gene. For example, binarytransformation vector 17146 (SEQ ID NO:33) contains an expressioncassette for HPPD variant (SEQ ID NO:24) linked with two PAT genecassettes (one with the 35S promoter and one with the CMP promoter, andboth PAT genes are followed by the nos terminator) for glufosinate basedselection during the transformation process. Another binarytransformation vector (17147) (SEQ ID NO:34) contains the HPPD variant(SEQ ID NO:24) expression cassette and also an EPSPS selectable markercassette. Vector 17147 was transformed into soybean and transgenicplants were obtained using glyphosate selection afterAgrobacterium-mediated transformation of immature seed targets.Similarly, binary vector 15764, (SEQ ID NO:35) was constructed tocomprise expression cassettes to express HPPD (SEQ ID NO:14) along witha bar selectable marker gene and binary vector 17149 (SEQ ID NO:36) wasconstructed to comprise an expression cassette expressing HPPD variant(SEQ ID NO:26) along with two PAT gene cassettes. In all cases the DNAsequences encoding the HPPD genes were codon-optimized for expression insoybean.

The Binary Vectors described above were constructed using a combinationof methods well known to those skilled in the art such as overlap PCR,DNA synthesis, restriction fragment sub-cloning and ligation. Theirunique structures are made explicit in FIGS. 6 (vector 17146), 7 (vector17147), 8 (vector 15764), and 9 (vector 17149), and in the sequencelistings (SEQ ID NOS:33-36). Additional information regarding thevectors shown in FIGS. 6-9 are provided below.

The abbreviations used in FIG. 6 (vector 17146) are defined as follows:

cAvHPPD-04

-   -   Start: 1024 End: 2343    -   Soybean codon optimized Oat HPPD gene encoding SEQ ID NO 24

cPAT-03-01

-   -   Start: 3209 End: 3760    -   PAT Hoescht A02774 synthetic S. viridochromogenes, plant codons;        identical to Q57146 phosphinothricin acetyl transferase protein

cPAT-03-02

-   -   Start: 5062 End: 5613    -   PAT Q57146 S. viridochromogenes phosphinothricin acetyl        transferase protein, cPAT-03-01 DNA, with mutated BamHI, Bg12        sites

cSpec-03

-   -   Start: 6346 End: 7134    -   Also called aadA; gene encoding the enzyme aminoglycoside 3′        adenyltransferase that confers resistance to spectinomycin and        streptomycin for maintenance of the vector in E. coli and        Agrobacterium. aka cSPEC-03

cVirG-01

-   -   Start: 7434 End: 8159    -   virG (putative) from pAD1289 with TTG start codon. virGN54D came        from pAD1289 described in Hansen et al. 1994, PNAS 91:7603-7607

cRepA-01

-   -   Start: 8189 End: 9262    -   RepA, pVS1 replication protein

eNOS-01

-   -   Start: 168 End: 259    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors.

eFMV-03

-   -   Start: 396 End: 589    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 596 End: 888    -   C to T & C to A by changes in Cauliflower mosaic virus 35S        enhancer region

eTMV-02

-   -   Start: 953 End: 1020    -   TMV Omega 5′UTR leader seq thought to enhance expression. EMBL:        TOTMV6

eFMV-03

-   -   Start: 4054 End: 4247    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 4254 End: 4546    -   C to T & C to A by changes in Cauliflower mosaic virus 35S        enhancer region

eNOS-01

-   -   Start: 4557 End: 4648    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors.

bNRB-05

-   -   Start: 4 End: 259 (Complementary)    -   Right border/NOS T-DNA region; may influence promoters. EMBL no:        701826, V00087, AF485783.

bN RB-01-01

-   -   Start: 101 End: 125 (Complementary)    -   Right Border Repeat of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bNLB-03

-   -   Start: 5937 End: 6066 (Complementary)    -   Left border region of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bN RB-01-01

-   -   Start: 5972 End: 5996 (Complementary)    -   25 bp Left border repeat region of T-DNA of Agrobacterium        tumefaciens nopaline ti-plasmid

prCMP-04

-   -   Start: 4655 End: 5051    -   Cestrum Yellow leaf curl virus promoter & leader (start        aagggagc?). genbank AF364175. US20040086447. prCMP-01 with 1        base pair truncation on 5′ end and 2 base pair truncation on 3′        end

pr35S-04-01

-   -   Start: 2664 End: 3184    -   35S promoter from Cauliflower Mosaic Virus. EMBL: CAMVG2

oVS1-02

-   -   Start: 9305 End: 9709    -   origin of replication and partitioning region from plasmid pVS1        of Pseudomonas (Itoh et al. 1984, Plasmid 11: 206-220); similar        to GenBank Accession Number U10487; serves as origin of        replication in Agrobacterium tumefaciens host

oCOLE-06

-   -   Start: 10387 End: 11193 (Complementary)    -   The ColE1 origin of replication functional in E. coli derived        from pUC19

tNOS-05-01

-   -   Start: 2360 End: 2612    -   synthetic Nopaline synthetase terminator

tNOS-05-01

-   -   Start: 3794 End: 4046    -   synthetic Nopaline synthetase terminator

tNOS-05-01

-   -   Start: 5642 End: 5894    -   synthetic Nopaline synthetase terminator

The abbreviations used in FIG. 7 (vector 17147) are defined as follows:

cAvHPPD-04

-   -   Start: 1024 End: 2343    -   Soybean codon optimized Oat HPPD gene encoding SEQ ID NO 24

cGmEPSPS-01

-   -   Start: 3672 End: 5249    -   Soybean codon-optimized version of double mutant soybean EPSPS        cDNA

cSpec-03

-   -   Start: 5982 End: 6770    -   Also called aadA; gene encoding the enzyme aminoglycoside 3′        adenyltransferase that confers resistance to spectinomycin and        streptomycin for maintenance of the vector in E. coli and        Agrobacterium. aka cSPEC-03

cVirG-01

-   -   Start: 7070 End: 7795    -   virG (putative) from pAD1289 with TTG start codon. virGN54D came        from pAD1289 described in Hansen et al. 1994, PNAS 91:7603-7607

cRepA-01

-   -   Start: 7825 End: 8898    -   RepA, pVS1 replication protein    -   Original Location Description:

eNOS-01

-   -   Start: 168 End: 259    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors.

eFMV-03

-   -   Start: 396 End: 589    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 596 End: 888    -   C to T & C to A by changes in Cauliflower mosaic virus 35S        enhancer region

eTMV-02

-   -   Start: 953 End: 1020    -   TMV Omega 5′UTR leader seq thought to enhance expression. EMBL:        TOTMV6

eFMV-03

-   -   Start: 2664 End: 2857    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 2864 End: 3156    -   C to T & C to A by changes in Cauliflower mosaic virus 35S        enhancer region

eNOS-01

-   -   Start: 3167 End: 3258    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors.

bNRB-05

-   -   Start: 4 End: 259 (Complementary)    -   Right border/NOS T-DNA region; may influence promoters. EMBL no:        J01826, V00087, AF485783.

bN RB-01-01

-   -   Start: 101 End: 125 (Complementary)    -   Right Border Repeat of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bNLB-03

-   -   Start: 5573 End: 5702 (Complementary)    -   Left border region of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bNLB-01-01

-   -   Start: 5608 End: 5632 (Complementary)    -   25 bp Left border repeat region of T-DNA of Agrobacterium        tumefaciens nopaline ti-plasmid

prCMP-04

-   -   Start: 3265 End: 3661    -   Cestrum Yellow leaf curl virus promoter & leader (start        aagggagc?). genbank AF364175. US20040086447. prCMP-01 with 1        base pair truncation on 5′ end and 2 base pair truncation on 3′        end    -   Original Location Description:

oVS1-02

-   -   Start: 8941 End: 9345    -   origin of replication and partitioning region from plasmid pVS1        of Pseudomonas (Itoh et al. 1984, Plasmid 11: 206-220); similar        to GenBank Accession Number U10487; serves as origin of        replication in Agrobacterium tumefaciens host

oCOLE-06

-   -   Start: 10023 End: 10829 (Complementary)    -   The ColE1 origin of replication functional in E. coli derived        from pUC19

tNOS-05-01

-   -   Start: 2360 End: 2612    -   synthetic Nopaline synthetase terminator

tNOS-05-01

-   -   Start: 5278 End: 5530    -   synthetic Nopaline synthetase terminator

The abbreviations used in FIG. 8 (vector 15764) are defined as follows:

cAvHPPD-03

-   -   Start: 450 End: 1769 (Complementary)    -   Soybean codon optimized Oat HPPD gene encoding SEQ ID NO 14

cPATBAR-07

-   -   Start: 3034 End: 3585    -   BAR X17220 S. hygroscopicus gene (mutated Bg12 site), caa35093        phosphinothricin acetyl transferase protein.

cSpec-03

-   -   Start: 4334 End: 5122    -   streptomycin adenylyltransferase; from Tn7 (aadA)

cVirG-01

-   -   Start: 5422 End: 6147    -   Virulence G gene from Agrobacterium tumefaciens(virGN54D,        containing TTG start codon) virGN54D came from pAD1289 described        in Hansen et al. 1994, PNAS 91:7603-7607

cRepA-03

-   -   Start: 6177 End: 7250    -   RepA, pVS1 replication protein with A to G at nt735

eTMV-02

-   -   Start: 1773 End: 1840 (Complementary)    -   Tobacco mosaic virus (TMV_Omega 5′UTR leader seq thought to        enhance expression.    -   EMBL: TOTMV6

e35S-05

-   -   Start: 1905 End: 2197 (Complementary)    -   Cauliflower mosaic virus 35S enhancer region with C to T & C to        A by changes.

eFMV-03

-   -   Start: 2204 End: 2397 (Complementary)    -   Figwort mosaic virus enhancer.

bNRB-04

-   -   Start: 5 End: 144 (Complementary)    -   Right border region of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid.    -   Differs from bNRB-03 by 20 by at 5′ end.

bN RB-01-01

-   -   Start: 102 End: 126 (Complementary)    -   Right Border Repeat of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid.

bNLB-03

-   -   Start: 3925 End: 4054 (Complementary)    -   Left border region of T-DNA from Agrobacterium tumefaciens        nopaline ti-plasmid. (Zambryski et al. 1980, Science,        209:1385-1391) EMBL no: J01825.

bNLB-01-01

-   -   Start: 3960 End: 3984 (Complementary)    -   25 bp Left border region of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid.

pr35S-04-01

-   -   Start: 2494 End: 3014    -   35S promoter; map originally defined promoter as 64 lbp long; no        exact match found in literature (LF July 2004)

oVS1-02

-   -   Start: 7293 End: 7697    -   origin of replication and partitioning region from plasmid pVS1        of Pseudomonas (Itoh et al. 1984, Plasmid 11: 206-220); similar        to GenBank Accession Number U10487; serves as origin of        replication in Agrobacterium tumefaciens host

oCOLE-06

-   -   Start: 8375 End: 9181 (Complementary)    -   ColE1 origin of replication functional in E. coli

tNOS-05-01

-   -   Start: 181 End: 433 (Complementary)    -   NOS terminator: 3′UTR of the nopaline synthase gene

tNOS-05-01

-   -   Start: 3619 End: 3871    -   NOS terminator: 3′UTR of the nopaline synthase gene

The abbreviations used in FIG. 9 (vector 17149) are defined as follows:

cAvHPPD-05

-   -   Start: 1024 End: 2343    -   Soybean codon optimized sequence encoding HPPD SEQ ID NO 26

cPAT-03-01

-   -   Start: 3209 End: 3760    -   PAT Hoescht A02774 synthetic S. viridochromogenes, plant codons;        identical to Q57146 phosphinothricin acetyl transferase protein

cPAT-03-02

-   -   Start: 5062 End: 5613    -   PAT Q57146 S. viridochromogenes phosphinothricin acetyl        transferase protein, cPAT-03-01 DNA, witht mutated BamH1, Bg12        sites

cSpec-03

-   -   Start: 6346 End: 7134    -   Also called aadA; gene encoding the enzyme aminoglycoside 3′        adenyltransferase that confers resistance to spectinomycin and        streptomycin for maintenance of the vector in E. coli and        Agrobacterium. aka cSPEC-03

cVirG-01

-   -   Start: 7434 End: 8159    -   virG (putative) from pAD1289 with TTG start codon. virGN54D came        from pAD1289 described in Hansen et al. 1994, PNAS 91:7603-7607

cRepA-01

-   -   Start: 8189 End: 9262    -   RepA, pVS1 replication protein    -   Original Location Description

eNOS-01

-   -   Start: 168 End: 259    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors.

eFMV-03

-   -   Start: 396 End: 589    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 596 End: 888    -   C to T & C to A by changes in Cauliflower mosaic virus 35S        enhancer region

eTMV-02

-   -   Start: 953 End: 1020    -   TMV Omega 5′UTR leader seq thought to enhance expression. EMBL:        TOTMV6

eFMV-03

-   -   Start: 4054 End: 4247    -   enhancer region from Figwort mosaic virus (FMV)

e35S-05

-   -   Start: 4254 End: 4546

eNOS-01

-   -   Start: 4557 End: 4648    -   Putative NOS enhancer sequence from 15235 as found in the right        border of certain binary vectors

bNRB-05

-   -   Start: 4 End: 259 (Complementary)    -   Right border/NOS T-DNA region; may influence promoters. EMBL no:        J01826, V00087, AF485783.

bNRB-01-01

-   -   Start: 101 End: 125 (Complementary)    -   Right Border Repeat of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bNLB-03

-   -   Start: 5937 End: 6066 (Complementary)    -   Left border region of T-DNA of Agrobacterium tumefaciens        nopaline ti-plasmid

bNLB-01-01

-   -   Start: 5972 End: 5996 (Complementary)    -   25 bp Left border repeat region of T-DNA of Agrobacterium        tumefaciens nopaline ti-plasmid

prCMP-04

-   -   Start: 4655 End: 5051    -   Cestrum Yellow leaf curl virus promoter & leader (start        aagggagc?). genbank AF364175. US20040086447. prCMP-01 with 1        base pair truncation on 5′ end and 2 base pair truncation on 3′        end

pr35S-04-01

-   -   Start: 2664 End: 3184    -   35s promoter from CaMV. EMBL: CAMVG2 (100% match against this        EMBL record)

oVS1-02

-   -   Start: 9305 End: 9709    -   origin of replication and partitioning region from plasmid pVS1        of Pseudomonas (Itoh et al. 1984, Plasmid 11: 206-220); similar        to GenBank Accession Number U10487; serves as origin of        replication in Agrobacterium tumefaciens host

oCOLE-06

-   -   Start: 10387 End: 11193 (Complementary)    -   The ColE1 origin of replication functional in E. coli derived        from pUC19

tNOS-05-01

-   -   Start: 2360 End: 2612    -   synthetic Nopaline synthetase terminator

tNOS-05-01

-   -   Start: 3794 End: 4046    -   synthetic Nopaline synthetase terminator

tNOS-05-01

-   -   Start: 5642 End: 5894    -   synthetic Nopaline synthetase terminator

Example 5 Transformation of Soybean and Selection of Herbicide-ResistantPlants

Soybean plant material can be suitably transformed and fertile plantsregenerated by many methods which are well known to one of skill in theart. For example, fertile morphologically normal transgenic soybeanplants may be obtained by: 1) production of somatic embryogenic tissuefrom, e.g., immature cotyledon, hypocotyl or other suitable tissue; 2)transformation by particle bombardment or infection with Agrobacterium;and 3) regeneration of plants. In one example, as described in U.S. Pat.No. 5,024,944, cotyledon tissue is excised from immature embryos ofsoybean, preferably with the embryonic axis removed, and cultured onhormone-containing medium so as to form somatic embryogenic plantmaterial. This material is transformed using, for example, direct DNAmethods, DNA coated microprojectile bombardment or infection withAgrobacterium, cultured on a suitable selection medium and regenerated,optionally also in the continued presence of selecting agent, intofertile transgenic soybean plants. Selection agents may be antibioticssuch as kanamycin, hygromycin, or herbicides such as phosphonothricin orglyphosate or, alternatively, selection may be based upon expression ofa visualisable marker gene such as GUS. Alternatively, target tissuesfor transformation comprise meristematic rather than somaclonalembryogenic tissue or, optionally, is flower or flower-forming tissue.Other examples of soybean transforamtions can be found, e.g. by physicalDNA delivery method, such as particle bombardment (Finer and McMullen(1991) In Vitro Cell Dev. Biol. 27P:175-182; McCabe et al. (1988)Bio/technology 6:923-926), whisker (Khalafalla et al. (2006) African J.of Biotechnology 5:1594-1599), aerosol bean injection (U.S. Pat. No.7,001,754), or by Agrobacterium-mediated delivery methods (Hinchee etal. (1988) Bio/Technology 6:915-922; U.S. Pat. No. 7,002,058; U.S.Patent App. Pub. No. 20040034889; U.S. Patent App. Pub. No. 20080229447;Paz et al. (2006) Plant Cell Report 25:206-213). The HPPD gene can alsobe delivered into organelle such as plastid to confer increasedherbicide resistance (U.S. Patent App. Pub. No. 20070039075).

Soybean transgenic plants can be generated with the above describedbinary vectors (Example 4) containing HPPD gene variants with differenttransformation methods. Optionally, the HPPD gene can provide the meansof selection and identification of transgenic tissue. For example, avector was used to transform immature seed targets as described (U.S.Patent App. Pub. No. 20080229447) to generate transgenic HPPD soybeanplants directly using HPPD inhibitor, such as mesotrione, as selectionagent. Optionally, HPPD genes can be present in the polynucleotidealongside other sequences which provide additional means ofselection/identification of transformed tissue including, for example,the known genes which provide resistance to kanamycin, hygromycin,phosphinothricin, butafenacil, or glyphosate. For example, differentbinary vectors containing PAT or EPSPS selectable marker genes asdescribed in Example 4 were transformed into immature soybean seedtarget to generate HPPD herbicide tolerant plants usingAgrobacterium-mediated transformation and glufosinate or glyphosateselection as described (U.S. Patent App. Pub. No. 20080229447).

Alternatively selectable marker sequences may be present on separatepolynucleotides and a process of, for example, co-transformation andco-selection is used. Alternatively, rather than a selectable markergene, a scorable marker gene such as GUS may be used to identifytransformed tissue.

An Agrobacterium-based method for soybean transformation can be used togenerate transgenic plants using glufosinate, glyphosate or HPPDinhibitor mesotrione as selection agent using immature soybean seeds asdescribed (U.S. Patent App. Pub. No. 20080229447).

Example 6 Soybean Transgenic Plant Growth, Analysis and HerbicideTolerance Evaluation

T0 plants were taken from tissue culture to the greenhouse where theywere transplanted into water-saturated soil (REDI-EARTH® Plug andSeedling Mix, Sun Gro Horticulture, Bellevue, Wash., or FafardGerminating Mix) mixed with 1% granular MARATHON® (Olympic HorticulturalProducts, Co., Mainland, Pa.) at 5-10 g/gal soil in 2″ square pots. Theplants were covered with humidty domes and placed in a Conviron chamber(Pembina, N. Dak.) with the following environmental conditions: 24° C.day; 20° C. night; 16-23 hr light-1-8 hrs dark photoperiod; 80% relativehumidity.

After plants became established in the soil and new growth appeared(˜1-2 weeks), plants were sampled and tested for the presence of desiredtransgene by TAQMAN® analysis using appropriate probes for the HPPDgenes, or promoters (for example prCMP). Positive plants weretransplanted into 4″ square pots containing Fafard #3 soil. Sierra17-6-12 slow release fertilizer was incorporated into the soil at therecommended rate. The plants were then relocated into a standardgreenhouse to acclimatize (˜1 week). The environmental conditions were:27° C. day; 21° C. night; 14 hr photoperiod (with supplemental light);ambient humidity. After acclimatizing (˜1 week), the plants were sampledand tested in detail for the presence and copy number of insertedtransgenes. Transgenic soybean plants were grown to maturity for T1 seedproduction. T1 plants were grown up, and after TAQMAN® analysis,homozygous plants were grown for seed production. Transgenic seeds andprogeny plants were used to further evaluate their herbicide toleranceperformance and molecular characteristics.

Homozygous soybean plants from 2 events made with vector 15764 (FIG. 8)and multiple events made with vector 17147 (FIG. 7) expressing SEQ IDNO:14 and SEQ ID NO:24, respectively, from identical HPPD expressioncassettes were grown and tested for tolerance to a range of HPPDherbicide. Table 8 summarises the results of these tests from plantssprayed at the V2 growth stage. Each data point represents the averagedamage score from n=7 replicates.

TABLE 8 Results of Herbicide Spray Tests Against Vector 15764 and 17147Soybean Events Chemical applied B C IFT E 420 840 400 420 368 g/ha g/hag/ha g/ha g/ha EVENT/ % % % % % HPPD SEQ dam. S.D. dam. S.D. dam. S.D.dam. S.D. dam. S.D. 1/SEQ#14 11.4 4.8 9.3 3.6 42.1 8.1 26.4 6.9 36.4 4.82/SEQ#14 20.7 3.4 22.1 3.9 52.9 9.9 42.5 4.2 52.1 7 S3/SEQ#24 15.3 2.415.3 3.7 62.1 6.4 30 4.1 51.4 6.3 T0/SEQ#24 8.3 2.1 5.3 2.1 45 4.1 19.35.3 39.3 11.7 S7/SEQ#24 10.6 2.4 6.9 2.4 45 4.1 20.7 3.4 41.4 3.8S8/SEQ#24 18.6 4.2 19.3 3.4 68.6 6.9 31.3 4.8 80 21.4 SF/SEQ#24 15.7 3.925 5.8 98.6 3.8 40 5.8 97.1 7.6 Jack w/t 82.9 8.6 83.6 4.8 82.1 3.9 96.13.5 84.3 8.4

Event 1 was most tolerant to mesotrione, and event 2 was the second mosttolerant 15764 event selected from a population of about ninety. Theseevents were used to compare the performance of five 17147 events. Fourof these, SF, S8, S7 and S3 had not been preselected for tolerance level(other than to confirm resistance, non-chimerical nature and thepresence of the gene) while the remaining event, TO, had beenpreselected as the most resistant out of five 17147 events in apreliminary field test.

Plants were in 4×4×4 inch plastic pots and grown under a 15/9 hour lightregime (daylight supplemented by artificial light in greenhouses) at aminimum night-time temperature of 18° C. and maximum daytime temperatureof 27° C. Soil was regular VBRC mix (1:1 mixture of Vero field soil andFafard Mix II) where Vero field soil is 98% sand and 2% clay. Treatmentswith compound B,=CALLISTO® 4 SC (480 g ai/L), with compound C (200 gai/L) EC, with IFT=Balance Pro 4 SC (480 g ai/L), and with compoundE=Laudis 3.5 SC (420 g ai/L) included 0.25% v/v INDUCE (a non-ionicsurfactant) and ammonium sulfate (N-PAK liquid AMS) at a rate equivalentto 8.5 lbs/gallon. Spray volume was 150 L/ha and the damage scoresreflect assessments at 14 DAT.

It is striking that, from such a small pool of 17147 events all fivetested provided tolerance to mesotrione and to isoxaflutole equivalentto one of the best 15764 events, event 2, and indeed that two of them,T0 and S7 exceed the performance of the most tolerant 15764 event, event1, that was selected from many.

The in vitro data, and in particular the off rate data, show that SEQ IDNO:24 is 2 and 2.3 fold superior to SEQ ID NO:14 in respect of B and IFTbut neutral in respect of C and E. In accord with this is the fact thatthe SEQ ID NO:24 HPPD expressing plants displayed a similarly alteredpattern of herbicide tolerance. Thus, for example, events SF and S8exhibits similar or better tolerance to both IFT and B than does 6W but,unlike 6W, essentially no tolerance to either compound E or C.Similarly, the only 17147 events, T0 and S7, to exhibit tolerance to Eand C that is close to that of event 4R also exhibit superior tolerancethan 4R to B and IFT. The in vitro data have predictive value in plantaand SEQ ID NO:24 provides improved tolerance to mesotrione and IFT butnot, for example, to tembotrione.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. (canceled)
 2. (canceled)
 3. A polynucleotide encoding a polypeptidehaving at least 759 sequence identity to SEQ ID NO:27, wherein saidpolypeptide has HPPD enzymatic activity and comprises the amino acidsequence GFGKGNFSE (SEQ ID NO:70), wherein the second G is replaced withany other amino acid.
 4. (canceled)
 5. (canceled)
 6. The polynucleotideof claim 3, wherein the nucleotide sequence is optimized for expressionin a plant.
 7. An expression cassette comprising the polynucleotide ofclaim 3 operably linked to a promoter that drives expression in a plantor plant cell.
 8. The expression cassette of claim 7 further comprisingan operably linked polynucleotide sequence encoding a polypeptide thatconfers a desirable trait.
 9. The expression cassette of claim 8,wherein said desirable trait is resistance or tolerance to an herbicide.10. The expression cassette of claim 9, wherein said desirable trait isresistance or tolerance to an HPPD inhibitor, glyphosate, orglufosinate.
 11. The expression cassette of claim 10, wherein saidpolypeptide that confers a desirable trait is a cytochrome P450 orvariant thereof.
 12. The expression cassette of claim 10, wherein saidpolypeptide that confers a desirable trait is an EPSPS(5-enol-pyrovyl-shikimate-3-phosphate-synthase).
 13. The expressioncassette of claim 10, wherein said polypeptide that confers a desirabletrait is a phosphinothricin acetyl transferase.
 14. A vector comprisingan expression cassette according to claim
 7. 15. The vector of claim 14,wherein said vector comprises a polynucleotide comprising the sequenceset forth in SEQ ID NO:33, 34, 35, or
 36. 16. A method for conferringresistance or tolerance to an HPPD inhibitor in a plant, said methodcomprising introducing into said plant at least one expression cassetteaccording to claim
 7. 17. A transformed plant cell comprising at leastone expression cassette according to claim
 7. 18. The plant cell ofclaim 17, wherein said plant cell is a rice, barley, potato, sweetpotato, canola, sunflower, rye, oats, wheat, corn, soybean, sugar beet,tobacco, Miscanthus grass, Switch grass, safflower, trees, cotton,cassaya, tomato, sorghum, alfalfa, sugar beet, and sugarcane plant cell.19. The plant cell of claim 18, wherein said plant cell is a soybeanplant cell.
 20. A plant, plant part, or seed comprising the plant cellof claim
 17. 21. A method of controlling weeds at a locus, said methodcomprising applying to said locus a weed-controlling amount of one ormore HPPD inhibitors, wherein said locus comprises a plant according toclaim
 20. 22. The method of claim 21, wherein said HPPD inhibitor isselected from the group consisting of: a) a compound of formula (Ia)

wherein R¹ and R² are hydrogen or together form an ethylene bridge; R³is hydroxy or phenylthio-; R⁴ is halogen, nitro, C₁-C₄ alkyl, C₁-C₄alkoxy-C₁-C₄ alkyl-, C₁-C₄ alkoxy-C₁-C₄ alkoxy-C₁-C₄ alkyl-; X ismethine, nitrogen, or C—R⁵ wherein R⁵ is hydrogen, C₁-C₄ alkoxy, C₁-C₄haloalkoxy-C₁-C₄ alkyl-, or a group

and R⁶ is C₁-C₄ alkylsulfonyl- or C₁-C₄ haloalkyl; b) a compound offormula (Ib)

R¹ and R² are independently C₁-C₄ alkyl; and the free acids thereof; c)a compound of formula (Ic)

wherein R¹ is hydroxy, phenylcarbonyl-C₁-C₄alkoxy- orphenylcarbonyl-C₁-C₄ alkoxy- wherein the phenyl moiety is substituted inpara-position by halogen or C₁-C₄ alkyl, or phenylsulfonyloxy- orphenylsulfonyloxy- wherein the phenyl moiety is substituted inpara-position by halogen or C₁-C₄ alkyl; R² is C₁-C₄ alkyl; R³ ishydrogen or C₁-C₄ alkyl; R⁴ and R⁶ are independently halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, or C₁-C₄ alkylsulfonyl-; and R⁵ is hydrogen,C₁-C₄alkyl, C₁-C₄ alkoxy-C₁-C₄alkoxy-, or a group

d) a compound of formula (Id)

wherein R¹ is hydroxy; R² is C₁-C₄ alkyl; R³ is hydrogen; and R⁴, R⁵ andR⁶ are independently C₁-C₄alkyl; e) a compound of formula (Ie)

wherein R¹ is cyclopropyl; R² and R⁴ are independently halogen,C₁-C₄haloalkyl, or C₁-C₄alkylsulfonyl-; and R³ is hydrogen; f) acompound of formula (If)

wherein R¹ is cyclopropyl; R² and R⁴ are independently halogen,C₁-C₄haloalkyl, or C₁-C₄alkylsulfonyl-; and R³ is hydrogen; and g) acompound of formula (Ig) or Formula (Ih)

wherein: — R² is selected from the group consisting of C₁-C₃alkyl,C₁-C₃-haloalkyl, C₁-C₃alkoxy-C₁-C₃alkyl andC₁-C₃alkoxy-C₂-C₃alkoxy-C₁-C₃-alkyl; R⁵ is hydrogen or methyl; R⁶ isselected from the group consisting of hydrogen, fluorine, chlorine,hydroxyl and methyl; R⁷ is selected from the group consisting ofhydrogen, halogen, hydroxyl, sulfhydryl, C₁-C₆alkyl, C₃-C₆cycloalkyl,C₁-C₆haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ alkenyl, C₃-C₆alkynyl,C₁-C₆alkoxy, C₄-C₇cycloalkoxy, C₁-C₆haloalkoxy, C₁-C₆alkylthio,C₁-C₆alkylsulfinyl, C₁-C₆alkylsulfonyl, C₁-C₆haloalkylthio, amino,C₁-C₆alkylamino, C₂-C₆dialkylamino, C₂-C₆dialkylaminosulfonyl,C₁-C₆alkylaminosulfonyl, C₁-C₆alkoxy-C₁-C₆alkyl,C₁-C₆alkoxy-C₂-C₆alkoxy, C₁-C₆alkoxy-C₂-C₆ alkoxy-C₁-C₆-alkyl,C₃-C₆alkenyl-C₂-C₆alkoxy, C₃-C₆alkynyl-C₁-C₆alkoxy C₁-C₆alkoxycarbonyl,C₁-C₅alkylcarbonyl, C₁-C₄alkylenyl-S(O)p-R′, C₁-C₄alkylenyl-CO₂—R′,C₁-C₄alkylenyl-(CO)N—R′R′, phenyl, phenylthio, phenylsulfinyl,phenylsulfonyl, phenoxy, pyrrolidinyl, piperidinyl, morpholinyl and 5 or6-membered heteroaryl or heteroaryloxy, the heteroaryl containing one tothree heteroatoms, each independently selected from the group consistingof oxygen, nitrogen and sulphur, wherein the phenyl or heteroarylcomponent may be optionally substituted by a substituent selected fromthe group consisting of C₁-C₃alkyl, C₁-C₃haloalkyl, C₁-C₃ alkoxy,C₁-C₃haloalkoxy, halo, cyano, and nitro; X=O or S; n=0 or 1; m=0 or 1with the proviso that if m=1 then n=0 and if n=1 then m=0; p=0, 1, or 2;R′ is independently selected from the group consisting of hydrogen andC₁-C₆alkyl; R⁸ is selected from the group consisting of hydrogen,C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkylcarbonyl-C₁-C₃alkyl,C₃-C₆cycloalkylalkeneyl for example cyclohexylmethylenyl,C₃-C₆alkynylalkyleneyl for example propargyl, C₂-C₅alkenylalkylenyl forexample allyl, C₁-C₆alkoxy C₁-C₆alkyl, cyano-C₁-C₆-alkyl,arylcarbonyl-C₁-C₃-alkyl (wherein the aryl may be optionally substitutedwith a substituent selected from the group consisting of halo,C₁-C₃-alkoxy, C₁-C₃-alkyl, C₁-C₃ haloalkyl), aryl-C₁-C₆alkyl (whereinthe aryl may be optionally substituted with a substituent selected fromthe group consisting of halo, C₁-C₃-alkoxy, C₁-C₃-alkyl, C₁-C₃haloalkyl), C₁-C₆alkoxy C₁-C₆alkoxy C₁-C₆alkyl and a 5 or 6-memberedheteroaryl-C₁-C₃-alkyl or heterocyclyl-C₁-C₃-alkyl, the heteroaryl orheterocyclyl containing one to three heteroatoms, each independentlyselected from the group consisting of oxygen, nitrogen and sulphur,wherein the heterocyclyl or heteroaryl component may be optionallysubstituted by a substituent selected from the group consisting of halo,C₁-C₃alkyl, C₁-C₃haloalkyl, and C₁-C₃ alkoxy; Q is selected from thegroup consisting of:

wherein A¹ is selected from the group consisting of O, C(O), S, SO, SO₂and (CR^(e)R^(f))_(q); q=0, 1 or 2; R^(a), R^(b), R^(c), R^(d), R^(e)and R^(f) are each independently selected from the group consisting ofC₁-C₄ alkyl which may be mono-, di- or tri-substituted by substituentsselected from the group consisting of C₁-C₄ alkoxy, halogen, hydroxy,cyano, hydroxycarbonyl, C₁-C₄ alkoxycarbonyl, C₁-C₄alkylthio,C₁-C₄alkylsulfinyl, C₁-C₄alkylsulfonyl, C₁-C₄ alkylcarbonyl, phenyl andheteroaryl, it being possible for the phenyl and heteroaryl groups inturn to be mono-, di- or tri-substituted by substituents selected fromthe group consisting of C₁-C₄alkoxy, halogen, hydroxy, cyano,hydroxycarbonyl, C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl andC₁-C₄haloalkyl, the substituents on the nitrogen in the heterocyclicring being other than halogen; or R^(a), R^(b), R^(c), R^(d), R^(e) andR^(f) are each independently selected from the group consisting ofhydrogen, C₁-C₄alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylthio, C₁-C₄alkylsulfinyl,C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl, phenyl or heteroaryl, it beingpossible for the phenyl and heteroaryl groups in turn to be mono-, di-or tri-substituted by substituents selected from the group consisting ofC₁-C₄ alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,C₁-C₄alkoxycarbonyl, C₁-C₄alkylsulfonyl and C₁-C₄haloalkyl, thesubstituents on the nitrogen in the heterocyclic ring being other thanhalogen; or R^(a) and R^(b) together form a 3- to 5-membered carbocyclicring which may be substituted by C₁-C₄alkyl and may be interrupted byoxygen, sulfur, S(O), SO₂, OC(O), NR^(g) or by C(O); or R^(a) and R^(c)together form a C₁-C₃alkylene chain which may be interrupted by oxygen,sulfur, SO, SO₂, OC(O), NR^(h) or by C(O); it being possible for thatC₁-C₃alkylene chain in turn to be substituted by C₁-C₄alkyl; R^(g) andR^(h) are each independently of the other C₁-C₄alkyl, C₁-C₄ haloalkyl,C₁-C₄alkylsulfonyl, C₁-C₄alkylcarbonyl or C₁-C₄alkoxycarbonyl; R^(i) isC₁-C₄alkyl; R³ is selected from the group consisting of C₁-C₆alkyl,optionally substituted with halogen and/or C₁-C₃alkoxy; and C₃-C₆cycloalkyl optionally substituted with halogen and/or C₁-C₃alkoxy; R⁹ isselected from the group consisting of cyclopropyl, CF₃ and i-Pr; R¹⁰ isselected from the group consisting of hydrogen, I, Br, SR¹¹, S(O)R¹¹,S(O)₂R¹¹; and R¹¹ is C₁₋₄ alkyl.
 23. The method of claim 22, whereinsaid HPPD inhibitor is mesotrione.
 24. The polynucleotide of claim 3,wherein the G is replaced with I in the encoded polypeptide.
 25. Thepolynucleotide of claim 3, wherein the G is replaced with A in theencoded polypeptide.
 26. The polynucleotide of claim 3, wherein the G isreplaced with A in the encoded polypeptide.